Modulators of beta-amyloid peptide aggregation

ABSTRACT

Compounds that modulate natural β amyloid peptide aggregation are provided. The modulators of the invention comprise a peptide, preferably based on a β amyloid peptide, that is comprised entirely of D-amino acids. Preferably, the peptide comprises 3-5 D-amino acid residues and includes at least two D-amino acid residues independently selected from the group consisting of D-leucine, D-phenylalanine and D-valine. In a particularly preferred embodiment, the peptide is a retro-inverso isomer of a β amyloid peptide, preferably a retro-inverso isomer of Aβ 17-21 . In certain embodiments, the peptide is modified at the amino-terminus, the carboxy-terminus, or both. Preferred amino-terminal modifying groups alkyl groups. Preferred carboxy-terminal modifying groups include an amide group, an acetate group, an alkyl amide group, an aryl amide group or a hydroxy group. Pharmaceutical compositions comprising the compounds of the invention, and diagnostic and treatment methods for amyloidogenic diseases using the compounds of the invention, are also disclosed.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional ApplicationNo. 60/122,736, filed on Mar. 4, 1999, incorporated herein in itsentirety by this reference.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD), first described by the Bavarianpsychiatrist Alois Alzheimer in 1907, is a progressive neurologicaldisorder that begins with short term memory loss and proceeds todisorientation, impairment of judgment and reasoning and, ultimately,dementia. The course of the disease usually leads to death in a severelydebilitated, immobile state between four and 12 years after onset. ADhas been estimated to afflict 5 to 11 percent of the population over age65 and as much as 47 percent of the population over age 85. The societalcost for managing AD is upwards of 80 billion dollars annually,primarily due to the extensive custodial care required for AD patients.Moreover, as adults born during the population boom of the 1940's and1950's approach the age when AD becomes more prevalent, the control andtreatment of AD will become an even more significant health careproblem. Currently, there is no treatment that significantly retards theprogression of the disease. For reviews on AD, see Selkoe, D. J. Sci.Amer., November 1991, pp. 68-78; and Yankner, B. A. et al. (1991) N.Eng. J. Med. 325:1849-1857.

[0003] It has recently been reported (Games et al. (1995) Nature373:523-527) that an Alzheimer-type neuropathology has been created intransgenic mice. The transgenic mice express high levels of human mutantamyloid precursor protein and progressively develop many of thepathological conditions associated with AD.

[0004] Pathologically, AD is characterized by the presence ofdistinctive lesions in the victim's brain. These brain lesions includeabnormal intracellular filaments called neurofibrillary tangles (NTFs)and extracellular deposits of amyloidogenic proteins in senile, oramyloid, plaques. Amyloid deposits are also present in the walls ofcerebral blood vessels of AD patients. The major protein constituent ofamyloid plaques has been identified as a 4 kilodalton peptide calledβ-amyloid peptide (β-AP)(Glenner, G. G. and Wong, C. W. (1984) Biochem.Biophys. Res. Commun. 120:885-890; Masters, C. et al. (1985) Proc. Natl.Acad. Sci. USA 82:4245-4249). Diffuse deposits of β-AP are frequentlyobserved in normal adult brains, whereas AD brain tissue ischaracterized by more compacted, dense-core β-amyloid plaques. (Seee.g., Davies, L. et al. (1988) Neurology 38:1688-1693) Theseobservations suggest that β-AP deposition precedes, and contributes to,the destruction of neurons that occurs in AD. In further support of adirect pathogenic role for β-AP, β-amyloid has been shown to be toxic tomature neurons, both in culture and in vivo. Yankner, B. A. et al.(1989) Science 245:417-420; Yankner, B. A. et al. (1990) Proc. Natl.Acad. Sci. USA 87:9020-9023; Roher, A. E. et al. (1991) Biochem.Biophys. Res. Commun. 174:572-579; Kowall, N. W. et al. (1991) Proc.Natl. Acad. Sci. USA 88:7247-7251. Furthermore, patients with hereditarycerebral hemorrhage with amyloidosis-Dutch-type (HCHWA-D), which ischaracterized by diffuse β-amyloid deposits within the cerebral cortexand cerebrovasculature, have been shown to have a point mutation thatleads to an amino acid substitution within β-AP. Levy, E. et al. (1990)Science 248:1124-1126. This observation demonstrates that a specificalteration of the β-AP sequence can cause β-amyloid to be deposited.

[0005] Natural β-AP is derived by proteolysis from a much larger proteincalled the amyloid precursor protein (APP). Kang, J. et al. (1987)Nature 325:733; Goldgaber, D. et al. (1987) Science 235:877; Robakis, N.K. et al. (1987) Proc. Natl. Acad. Sci. USA 84:4190; Tanzi, R. E. et al.(1987) Science 235:880. The APP gene maps to chromosome 21, therebyproviding an explanation for the β-amyloid deposition seen at an earlyage in individuals with Down's syndrome, which is caused by trisomy ofchromosome 21. Mann, D. M. et al. (1989) Neuropathol. Appl. Neurobiol.15:317; Rumble, B. et al. (1989) N. Eng. J. Med. 320:1446. APP containsa single membrane spanning domain, with a long amino terminal region(about two-thirds of the protein) extending into the extracellularenvironment and a shorter carboxy-terminal region projecting into thecytoplasm. Differential splicing of the APP messenger RNA leads to atleast five forms of APP, composed of either 563 amino acids (APP-563),695 amino acids (APP-695), 714 amino acids (APP-714), 751 amino acids(APP-751) or 770 amino acids (APP-770).

[0006] Within APP, naturally-occurring β amyloid peptide begins at anaspartic acid residue at amino acid position 672 of APP-770.Naturally-occurring β-AP derived from proteolysis of APP is 39 to 43amino acid residues in length, depending on the carboxy-terminal endpoint, which exhibits heterogeneity. The predominant circulating form ofβ-AP in the blood and cerebrospinal fluid of both AD patients and normaladults is β1-40 (“short β”). Seubert, P. et al. (1992) Nature 359:325;Shoji, M. et al. (1992) Science 258:126. However, β1-42 and β1-43 (“longβ”) also are forms in β-amyloid plaques. Masters, C. et al. (1985) Proc.Natl. Acad. Sci. USA 82:4245; Miller, D. et al. (1993) Arch. Biochem.Biophys. 301:41; Mori, H. et al. (1992) J. Biol. Chem. 267:17082.Although the precise molecular mechanism leading to β-APP aggregationand deposition is unknown, the process has been likened to that ofnucleation-dependent polymerizations, such as protein crystallization,microtubule formation and actin polymerization. See e.g., Jarrett, J. T.and Lansbury, P. T. (1993) Cell 73:1055-1058. In such processes,polymerization of monomer components does not occur until nucleusformation. Thus, these processes are characterized by a lag time beforeaggregation occurs, followed by rapid polymerization after nucleation.Nucleation can be accelerated by the addition of a “seed” or preformednucleus, which results in rapid polymerization. The long β forms of β-APhave been shown to act as seeds, thereby accelerating polymerization ofboth long and short β-AP forms. Jarrett, J. T. et al. (1993)Biochemistry 32:4693.

[0007] In one study, in which amino acid substitutions were made inβ-AP, two mutant β peptides were reported to interfere withpolymerization of non-mutated β-AP when the mutant and non-mutant formsof peptide were mixed. Hilbich, C. et al. (1992) J. Mol. Biol.228:460-473. Equimolar amounts of the mutant and non-mutant (i.e.,natural) β amyloid peptides were used to see this effect and the mutantpeptides were reported to be unsuitable for use in vivo. Hilbich, C. etal. (1992), supra.

SUMMARY OF THE INVENTION

[0008] This invention pertains to compounds, and pharmaceuticalcompositions thereof, that can bind to natural β amyloid peptides(β-AP), modulate the aggregation of natural β-AP and/or inhibit theneurotoxicity of natural β-APs. The compounds are modified in a mannerwhich allows for increased biostability and prolonged elevated plasmalevels. The β-amyloid modulator compounds of the invention comprise apeptidic structure, preferably based on β-amyloid peptide, that iscomposed entirely of D-amino acids. In various embodiments, the peptidicstructure of the modulator compound comprises a D-amino acid sequencecorresponding to a L-amino acid sequence found within natural β-AP, aD-amino acid sequence which is an inverso isomer of an L-amino acidsequence found within natural β-AP, a D-amino acid sequence which is aretro-inverso isomer of an L-amino acid sequence found within naturalβ-AP, or a D-amino acid sequence that is a scrambled or substitutedversion of an L-amino acid sequence found within natural β-AP.Preferably, the D-amino acid peptidic structure of the modulator isdesigned based upon a subregion of natural β-AP at positions 17-21(Aβ₁₇₋₂₀ and Aβ₁₇₋₂₁, respectively), which has the amino acid sequencesLeu-Val-Phe-Phe-Ala (SEQ ID NO:4). In preferred embodiments, aphenylalanine in the compounds of the invention is. substituted with aphenylalanine analogue which is more stable and less prone to, forexample, oxidative metabolism, or allows for increased brain levels ofthe compound.

[0009] In yet another embodiment, a modulator compound of the inventionincludes a β-amyloid peptide comprised of D-amino acids, L-amino acidsor both, an inverso isomer of a β-amyloid peptide, or a retro-inversoisomer of a β-amyloid peptide which is attached to a hydrazine moiety,wherein the compound binds to natural β-amyloid peptides or modulatesthe aggregation or inhibits the neurotoxicity of natural β-amyloidpeptides when contacted with the natural β-amyloid peptides.

[0010] A modulator compound of the invention preferably comprises 3-20D-amino acids, more preferably 3-10 D-amino acids and even morepreferably 3-5 D-amino acids. The D-amino acid peptidic structure of themodulator can have free amino-, carboxy-, or carboxy amide-termini.Alternatively, the amino-terminus, the carboxy-terminus or both may bemodified. For example, an N-terminal modifying group can be used thatenhances the ability of the compound to inhibit Aβ aggregation.Moreover, the amino- and/or carboxy termini of the peptide can bemodified to alter a pharmacokinetic property of the compound (such asstability, bioavailability, e.g., enhanced delivery of the compoundacross the blood brain barrier and entry into the brain, and the like).Preferred amino-terminal modifying groups include alkyl groups, e.g.,methyl, ethyl, or isopropyl groups. Preferred carboxy-terminal modifyinggroups include amide groups, alkyl or aryl amide groups (e.g.,phenethylamide), hydroxy groups (i.e., reduction products of peptideacids, resulting in peptide alcohols), acyl amide groups, and acetylgroups. Still further, a modulator compound can be modified to label thecompound with a detectable substance (e.g., a radioactive label).

[0011] In certain preferred embodiments, the invention provides acompound having the structure:N,N-dimethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-dimethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-methyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-ethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-isopropyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-isopropylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-dimethylamide;N,N-diethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-diethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-ethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-ethyl-(Gly-D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-methyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-ethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-propyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-diethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ile)-NH₂;H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ala-)-NH₂;H-(D-Ile-D-Ile-D-Phe-D-Phe-D-Ile)-NH₂;H-(D-Nle-D-Val-D-Phe-D-Phe-D-Ala-)-NH₂;H-(D-Nle-D-Val-D-Phe-D-Phe-D-Nle)-NH₂;1-piperidine-acetyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;1-piperidine-acetyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-D-Leu-D-Val-D-Phe-D-Phe-D-Leu-isopropylamide;H-D-Leu-D-Phe-D-Phe-D-Val-D-Leu-isopropylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-methylamide;H-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-methylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-OH;N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-[F₅]Phe-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-Cha-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-[p-F]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-[F₅]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-Lys-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Cha-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[p-F]Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[F₅]Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Lys-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Cha-D-Cha-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[p-F]Phe-D-[p-F]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[F₅]Phe-D-[F₅]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Lys-D-Lys-D-Val-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-[F₅]Phe-D-Leu)-NH₂;H-D-Leu-D-Val-D-Phe-NH-(H-D-Leu-D-Val-D-Phe-)NH;H-D-Leu-D-Val-D-Phe-NH-NH-COCH₃; and H-D-Leu-D-Val-D-Phe-NH-NH₂.

[0012] Particularly preferred compounds of the invention are set forthin the Examples.

[0013] Another aspect of the invention pertains to pharmaceuticalcompositions. Typically, the pharmaceutical composition comprises atherapeutically effective amount of a modulator compound of theinvention and a pharmaceutically acceptable carrier.

[0014] Yet another aspect of the invention pertains to methods forinhibiting aggregation of natural β-amyloid peptides. These methodscomprise contacting the natural β-amyloid peptides with a modulatorcompound of the invention such that aggregation of the natural β-amyloidpeptides is inhibited.

[0015] Yet another aspect of the invention pertains to methods fordetecting the presence or absence of natural β-amyloid peptides in abiological sample. These methods comprise contacting a biological samplewith a compound of the invention, wherein the compound is labeled with adetectable substance, and detecting the compound bound to naturalβ-amyloid peptides to thereby detect the presence or absence of naturalβ-amyloid peptides in the biological sample.

[0016] Still another aspect of the invention pertains to methods fortreating a subject for a disorder associated with β-amyloidosis. Thesemethods comprise administering to the subject a therapeuticallyeffective amount of a modulator compound of the invention such that thesubject is treated for a disorder associated with β-amyloidosis.Preferably, the disorder is Alzheimer's disease. Use of the modulatorsof the invention for therapy or for the manufacture of a medicament forthe treatment of a disorder associated with β-amyloidosis is alsoencompassed by the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a table depicting the results from a brain uptake assay.

[0018]FIG. 2 is a graph depicting the results from the fibril bindingassay described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

[0019] This invention pertains to compounds, and pharmaceuticalcompositions thereof, that can bind to natural β-amyloid peptides,modulate the aggregation of natural β amyloid peptides (β-AP) and/orinhibit the neurotoxicity of natural β-APs. The compounds are modifiedin a manner which allows for increased biostability and prolongedelevated plasma levels. A compound of the invention that modulatesaggregation of natural β-AP, referred to herein interchangeably as a βamyloid modulator compound, a β amyloid modulator or simply a modulator,alters the aggregation of natural β-AP when the modulator is contactedwith natural β-AP. Thus, a compound of the invention acts to alter thenatural aggregation process or rate for β-AP, thereby disrupting thisprocess. Preferably, the compounds inhibit β-AP aggregation. Thecompounds of the invention are characterized in that they comprise apeptidic structure composed entirely of D-amino acid residues. Thispeptidic structure is preferably based on β-amyloid peptide and cancomprise, for example, a D-amino acid sequence corresponding to aL-amino acid sequence found within natural β-AP, a D-amino acid sequencewhich is an inverso isomer of an L-amino acid sequence found withinnatural β-AP, a D-amino acid sequence which is a retro-inverso isomer ofan L-amino acid sequence found within natural β-AP, or a D-amino acidsequence that is a scrambled or substituted version of an L-amino acidsequence found within natural β-AP. In preferred embodiments, thephenylalanines in the compounds of the invention are substituted withphenylalanine analogues which are more stable and less prone to, forexample, oxidative metabolism.

[0020] The invention encompasses modulator compounds comprising aD-amino acid peptidic structure having free amino-, carboxy-, or carboxyamide-termini, as well as modulator compounds in which theamino-terminus, the carboxy-terminus, and/or side chain(s) of thepeptidic structure are modified.

[0021] The β amyloid modulator compounds of the invention can beselected based upon their ability to bind to natural β-amyloid peptides,modulate the aggregation of natural β-AP in vitro and/or inhibit theneurotoxicity of natural β-AP fibrils for cultured cells (using assaysdescribed herein, for example, the neurotoxicity assay, the nucleationassay, or the fibril binding assay). Preferred modulator compoundsinhibit the aggregation of natural β-AP and/or inhibit the neurotoxicityof natural β-AP. However, modulator compounds selected based on one orboth of these properties may have additional properties in vivo that maybe beneficial in the treatment of amyloidosis (J. S. Pachter et al.(1998) “Aβ1-40 induced neurocytopathic activation of human monocytes isblocked by Aβ peptide aggregation inhibitors. ” Neurobiology of Aging(Abstracts: The 6^(th) International Conference on Alzheimer's Diseaseand Related Disorders, Amsterdam, 18-23 July 1998) 19, S 128 (Abstract540); R. Weltzein, A. et al. (1998) “Phagocytosis of Beta-Amyloid: APossible Requisite for Neurotoxicity.” J. Neuroimmunology (SpecialIssue: Abstracts of the International Society of Neuroimmunology FifthInternational Congress, Montreal, Canada, 23-27 August 1998) 1998, 90,32 (Abstract 162)). For example, the modulator compound may interferewith processing of natural β-AP (either by direct or indirect proteaseinhibition) or by modulation of processes that produce toxic β-AP, orother APP fragments, in vivo. Alternatively, modulator compounds may beselected based on these latter properties, rather than inhibition of Aβaggregation in vitro. Moreover, modulator compounds of the inventionthat are selected based upon their interaction with natural β-AP alsomay interact with APP or with other APP fragments. Still further, amodulator compound of the invention can be characterized by its abilityto bind to β-amyloid fibrils (which can be determined, for example, byradiolabeling the compound, contacting the compound with β-amyloidplaque and counting or detecting, e.g., by imaging, the compound boundto pathological forms of β-AP, e.g., the plaque), while notsignificantly altering the aggregation of the β-amyloid fibrils. Such acompound that binds efficiently to β-amyloid fibrils while notsignificantly altering the aggregation of the β-amyloid fibrils can beused, for example, to detect β-amyloid fibrils (e.g., for diagnosticpurposes, as described further herein). It should be appreciated,however, that the ability of a particular compound to bind to β-amyloidfibrils and/or modulate their aggregation may vary depending upon theconcentration of the compound. Accordingly, a compound that, at a lowconcentration, binds to β-amyloid fibrils without altering theiraggregation may nevertheless inhibit aggregation of the fibrils at ahigher concentration. All such compounds having the property of bindingto β-amyloid fibrils and/or modulating the aggregation of the fibrilsare intended to be encompassed by the invention.

[0022] As used herein, a “modulator” of β-amyloid aggregation isintended to refer to an agent that, when contacted with natural βamyloid peptides, alters the aggregation of the natural β amyloidpeptides. The term “aggregation of β amyloid peptides” refers to aprocess whereby the peptides associate with each other to form amultimeric, largely insoluble complex. The term “aggregation” further isintended to encompass β amyloid fibril formation and also encompassesβ-amyloid plaques.

[0023] The terms “natural β-amyloid peptide”, “natural β-AP” and“natural Aβ peptide”, used interchangeably herein, are intended toencompass naturally occurring proteolytic cleavage products of the βamyloid precursor protein (APP) which are involved in β-AP aggregationand β-amyloidosis. These natural peptides include β-amyloid peptideshaving 39-43 amino acids (i.e., Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁, Aβ₁₋₄₂ andAβ₁₋₄₃). The amino-terminal amino acid residue of natural β-APcorresponds to the aspartic acid residue at position 672 of the 770amino acid residue form of the amyloid precursor protein (“APP-770”).The 43 amino acid long form of natural β-AP has the amino acid sequenceDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT (also shown in SEQ ID NO:1),whereas the shorter forms have 1-4 amino acid residues deleted from thecarboxy-terminal end. The amino acid sequence of APP-770 from position672 (i.e., the amino-terminus of natural β-AP) to its C-terminal end(103 amino acids) is shown in SEQ ID NO:2. The preferred form of naturalβ-AP for use in the aggregation assays described herein is Aβ₁₋₄₀ orAβ₁₋₄₂.

[0024] In the presence of a modulator of the invention, aggregation ofnatural β amyloid peptides is “altered” or “modulated”. The variousforms of the term “alteration” or “modulation” are intended to encompassboth inhibition of β-AP aggregation and promotion of β-AP aggregation.Aggregation of natural β-AP is “inhibited” in the presence of themodulator when there is a decrease in the amount and/or rate of β-APaggregation as compared to the amount and/or rate of β-AP aggregation inthe absence of the modulator. The various forms of the term “inhibition”are intended to include both complete and partial inhibition of β-APaggregation. Inhibition of aggregation can be quantitated as the foldincrease in the lag time for aggregation or as the decrease in theoverall plateau level of aggregation (i.e.,. total amount ofaggregation), using an aggregation assay as described in the Examples.In various embodiments, a modulator of the invention increases the lagtime of aggregation at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold,2.5-fold, 3-fold, 4-fold or 5-fold, for example, when the compound is ata one molar equivalent to the β-AP. In various other embodiments, amodulator of the invention inhibits the plateau level of aggregation atleast 10%, 20%, 30%, 40%, 50%, 75% or 100%.

[0025] A modulator which inhibits β-AP aggregation (an “inhibitorymodulator compound”) can be used to prevent or delay the onset ofβ-amyloid deposition. Preferably, inhibitory modulator compounds of theinvention inhibit the formation and/or activity of neurotoxic aggregatesof natural Aβ peptide (i.e., the inhibitory compounds can be used toinhibit the neurotoxicity of β-AP). Additionally, the inhibitorycompounds of the invention can reduce the neurotoxicity of preformedβ-AP aggregates, indicating that the inhibitory modulators can eitherbind to preformed Aβ fibrils or soluble aggregate and modulate theirinherent neurotoxicity or that the modulators can perturb theequilibrium between monomeric and aggregated forms of β-AP in favor ofthe non-neurotoxic form.

[0026] Alternatively, in another embodiment, a modulator compound of theinvention promotes the aggregation of natural Aβ peptides. The variousforms of the term “promotion” refer to an increase in the amount and/orrate of β-AP aggregation in the presence of the modulator, as comparedto the amount and/or rate of β-AP aggregation in the absence of themodulator. Such a compound which promotes Aβ aggregation is referred toas a stimulatory modulator compound. Stimulatory modulator compounds maybe useful for sequestering β-amyloid peptides, for example in abiological compartment where aggregation of β-AP may not be deleteriousto thereby deplete β-AP from a biological compartment where aggregationof β-AP is deleterious. Moreover, stimulatory modulator compounds can beused to promote Aβ aggregation in in vitro aggregation assays (e.g.,assays such as those described in Example 2), for example in screeningassays for test compounds that can then inhibit or reverse this Aβaggregation (i.e., a stimulatory modulator compound can act as a “seed”to promote the formation of AP aggregates).

[0027] In a preferred embodiment, the modulators of the invention arecapable of altering β-AP aggregation when contacted with a molar excessamount of natural β-AP. A “molar excess amount of natural β-AP” refersto a concentration of natural β-AP, in moles, that is greater than theconcentration, in moles, of the modulator. For example, if the modulatorand β-AP are both present at a concentration of 1 μM, they are said tobe “equimolar”, whereas if the modulator is present at a concentrationof 1 μM and the β-AP is present at a concentration of 5 μM, the β-AP issaid to be present at a 5-fold molar excess amount compared to themodulator. In preferred embodiments, a modulator of the invention iseffective at altering natural β-AP aggregation when the natural β-AP ispresent at at least a 2-fold, 3-fold or 5-fold molar excess compared tothe concentration of the modulator. In other embodiments, the modulatoris effective at altering β-AP aggregation when the natural β-AP ispresent at at least a 10-fold, 20-fold, 33-fold, 50-fold, 100-fold,500-fold or 1000-fold molar excess compared to the concentration of themodulator.

[0028] As used herein, the term “β amyloid peptide comprised entirely ofD-amino acids”, as used in a modulator of the invention, is intended toencompass peptides having an amino acid sequence identical to that ofthe natural sequence in APP, as well as peptides having acceptable aminoacid substitutions from the natural sequence, but which is composed ofD-amino acids rather than the natural L-amino acids present in naturalβ-AP. Acceptable amino acid substitutions are those that do not affectand/or may improve the ability of the D-amino acid-containing peptide toalter natural β-AP aggregation. Moreover, particular amino acidsubstitutions may further contribute to the ability of the peptide toalter natural β-AP aggregation and/or may confer additional beneficialproperties on the peptide (e.g., increased solubility, reducedassociation with other amyloid proteins, etc.). A peptide having anidentical amino acid sequence to that found within a parent peptide butin which all L-amino acids have been substituted with all D-amino acidsis also referred to as an “inverso” compounds. For example, if a parentpeptide is Thr-Ala-Tyr, the inverso form is D-Thr-D-Ala-D-Tyr.

[0029] As used herein, the term “retro-inverso isomer of a β amyloidpeptide”, as used in a modulator of the invention, is intended toencompass peptides in which the sequence of the amino acids is reversedas compared to the sequence in natural β-AP and all L-amino acids arereplaced with D-amino acids. For example, if a parent peptide isThr-Ala-Tyr, the retro-inverso form is D-Tyr-D-Ala-D-Thr. Compared tothe parent peptide, a retro-inverso peptide has a reversed backbonewhile retaining substantially the original spatial conformation of theside chains, resulting in a retro-inverso isomer with a topology thatclosely resembles the parent peptide. See Goodman et al “Perspectives inPeptide Chemistry” pp. 283-294 (1981). See also U.S. Pat. No. 4,522,752by Sisto for further description of “retro-inverso” peptides.

[0030] Various additional aspects of the modulators of the invention,and the uses thereof, are described in further detail in the followingsubsections.

[0031] I. Modulator Compounds

[0032] In one embodiment, a modulator compound of the inventioncomprises a β-amyloid peptide, the β-amyloid peptide being comprisedentirely of D-amino acids, wherein the compound binds to naturalβ-amyloid peptides or modulates the aggregation or inhibits theneurotoxicity of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. Preferably, the β-amyloid peptide of themodulator is comprised of 3-20 D-amino acids, more preferably 3-10D-amino acids, and even more preferably 3-5 D-amino acids. In preferredembodiments, a phenylalanine in the compounds of the invention issubstituted with a phenylalanine analogue which is more stable and lessprone to, for example, oxidative metabolism.

[0033] In one embodiment, the β-amyloid peptide of the modulator isamino-terminally modified, for example, with a modifying groupcomprising an alkyl group such as a C1-C6 lower alkyl group, e.g., amethyl, ethyl, or propyl group; or a cyclic, heterocyclic, polycyclic orbranched alkyl group. Examples of suitable N-terminal modifying groupsare described further in subsection II below. In another embodiment, theβ-amyloid peptide of the modulator is carboxy-terminally modified, forexample the modulator can comprise a peptide amide, a peptide alkyl oraryl amide (e.g., a peptide phenethylamide) or a peptide alcohol.Examples of suitable C-terminal modifying groups are described furtherin subsections II and III below. The β-amyloid peptide of the modulatormay be modified to enhance the ability of the modulator to alter β-APaggregation or neurotoxicity. Additionally or alternatively, β-amyloidpeptide of the modulator may be modified to alter a pharmacokineticproperty of the modulator and/or to label the modulator with adetectable substance (described further in subsection III below).

[0034] In another embodiment, a modulator compound of the inventioncomprises a retro-inverso isomer of a β-amyloid peptide, wherein thecompound binds to natural β-amyloid peptides or modulates theaggregation or inhibits the neurotoxicity of natural β-amyloid peptideswhen contacted with the natural β-amyloid peptides. Preferably, theretro-inverso isomer of the β-amyloid peptide is comprised of 3-20D-amino acids, more preferably 3-10 D-amino acids, and even morepreferably 3-5 D-amino acids. In preferred embodiments, thephenylalanines in the compounds of the invention are substituted withphenylalanine analogues which are more stable and less prone to, forexample, oxidative metabolism.

[0035] In one embodiment, the retro-inverso isomer is amino-terminallymodified, for example, with a modifying group comprising an alkyl groupsuch as a C1-C6 lower alkyl group, e.g., a methyl, ethyl, or propylgroup; or a cyclic, heterocyclic, polycyclic or branched alkyl group.Examples of suitable N-terminal modifying groups are described furtherin subsection II below. In another embodiment, the retro-inverso isomeris carboxy-terminally modified, for example with an amide group, analkyl or aryl amide group (e.g., phenethylamide) or a hydroxy group(i.e., the reduction product of a peptide acid, resulting in a peptidealcohol). Examples of suitable C-terminal modifying groups are describedfurther in subsections II and III below. The retro-inverso isomer may bemodified to enhance the ability of the modulator to alter β-APaggregation or neurotoxicity. Additionally or alternatively, theretro-inverso isomer may be modified to alter a pharmacokinetic propertyof the modulator and/or to label the modulator with a detectablesubstance (described further in subsection III below).

[0036] In yet another embodiment, a modulator compound of the inventionincludes a β-amyloid peptide comprised entirely or partially of D-aminoacids, an inverso isomer of a β-amyloid peptide, or a retro-inversoisomer of a β-amyloid peptide which is attached to a hydrazine moiety,wherein the compound binds to natural β-amyloid peptides or modulatesthe aggregation or inhibits the neurotoxicity of natural β-amyloidpeptides when contacted with the natural β-amyloid peptides. Preferably,the modulator compound of the invention is comprised of 1-20 D-aminoacids, more preferably 1-10 D-amino acids, even more preferably 1-5D-amino acids, and most preferably 2-4 D-amino acids which are attachedto a hydrazine moiety.

[0037] In one embodiment, the modulator compounds of the invention whichinclude a hydrazine moiety are amino-terminally modified, for examplewith a modifying comprising an alkyl group, e.g., a methyl, ethyl, orisopropyl group. Examples of suitable N-terminal modifying groups aredescribed further in subsection II below. In another embodiment,modulator compounds of the invention which include a hydrazine moietyare carboxy-terminally modified, for example with an acetyl. Examples ofsuitable C-terminal modifying groups are described further insubsections II and III below. The modulator compounds of the inventionwhich include a hydrazine moiety may be modified to enhance the abilityof the modulator to alter β-AP aggregation or neurotoxicity.Additionally or alternatively, the modulator compounds of the inventionwhich include a hydrazine moiety may be modified to alter apharmacokinetic property of the modulator and/or to label the modulatorwith a detectable substance (described further in subsection III below).

[0038] The modulators of the invention preferably are designed basedupon the amino acid sequence of a subregion of natural β-AP. The term“subregion of a natural β-amyloid peptide” is intended to includeamino-terminal and/or carboxy-terminal deletions of natural β-AP. Theterm “subregion of natural β-AP” is not intended to include full-lengthnatural β-AP (ie., “subregion” does not include Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁,Aβ₁₋₄₂ and Aβ₁₋₄₃). A preferred subregion of natural β-amyloid peptideis an “Aβ aggregation core domain” (ACD). As used herein, the term “Aβaggregation core domain” refers to a subregion of a natural β-amyloidpeptide that is sufficient to modulate aggregation of natural β-APs whenthis subregion, in its L-amino acid form, is appropriately modified(e.g., modified at the amino-terminus), as described in detail in U.S.patent application Ser. No. 08/548,998 and U.S. patent application Ser.No. 08/616,081, the entire contents of each of which are expresslyincorporated herein by reference. Preferably, the ACD is modeled after asubregion of natural β-AP that is less than 15 amino acids in length andmore preferably is between 3-10 amino acids in length. In variousembodiments, the ACD is modeled after a subregion of β-AP that is 10, 9,8, 7, 6, 5, 4 or 3 amino acids in length. In one embodiment, thesubregion of β-AP upon which the ACD is modeled is an internal orcarboxy-terminal region of β-AP (i.e., downstream of the amino-terminusat amino acid position 1). In another embodiment, the ACD is modeledafter a subregion of β-AP that is hydrophobic.

[0039] Preferred Aβ aggregation core domains encompass amino acidresidues 17-20 or 17-21 of natural β-AP (Aβ₁₇₋₂₀ and Aβ₁₇₋₂₁,respectively) and analogues thereof, as described herein. The amino acidsequences of Aβ₁₇₋₂₀ and Aβ₁₇₋₂₁ are Leu-Val-Phe-Phe (SEQ ID NO:3) andLeu-Val-Phe-Phe-Ala (SEQ ID NO:4), respectively.

[0040] As demonstrated in the Examples, D-amino acid-containingmodulators designed based upon the amino acid sequences of Aβ₁₇₋₂₀ andAβ₁₇₋₂₁ are particularly effective inhibitors of Aβ aggregation andexhibit an enhanced biostability and prolonged elevated plasma levels.These modulators can comprise a D-amino acid sequence corresponding tothe L-amino acid sequence of Aβ₁₇₋₂₀ or Aβ₁₇₋₂₁, a D-amino acid sequencewhich is an inverso isomer of the L-amino acid sequence of Aβ₁₇₋₂₀ orAβ₁₇₋₂₁, a D-amino acid sequence which is a retro-inverso isomer of theL-amino acid sequence of Aβ₁₇₋₂₀or Aβ₁₇₋₂₁, or a D-amino acid sequencethat is a scrambled or substituted version of the L-amino acid sequenceof Aβ₁₇₋₂₀ or Aβ₁₇₋₂₁. In preferred embodiments, a phenylalanine in themodulators designed based upon the amino acid sequences of Aβ₁₇₋₂₀ andAβ₁₇₋₂ is substituted with a phenylalanine analogue which is more stableand less prone to, for example, oxidative metabolism. In other preferredembodiments, the modulators designed based upon the amino acid sequencesof Aβ₁₇₋₂₀ and Aβ₁₇₋₂ further comprise a hydrazine moiety.

[0041] The D-amino acid-based modulators may have unmodified amino-and/or carboxy-termini and/or carboxy amide termini, or, alternatively,the amino-terminus, the carboxy-terminus, or both, may be modified(described further below). The peptidic structures of effectivemodulators generally are hydrophobic and are characterized by thepresence of at least two D-amino acid structures independently selectedfrom the group consisting of a D-leucine structure, a D-phenylalaninestructure and a D-valine structure. As used herein, the term a “D-aminoacid structure” (such as a “D-leucine structure”, a “D-phenylalaninestructure” or a “D-valine structure”) is intended to include the D-aminoacid, as well as analogues, derivatives and mimetics of the D-amino acidthat maintain the functional activity of the compound (discussed furtherbelow). For example, the term “D-phenylalanine structure” is intended toinclude D-phenylalanine as well as D-cyclohexylalanine [D-cha],D-4-fluorophenylalanine (para-fluorophenylalanine) {[p-F]f orD-[p-F]Phe}, D-pentafluorophenylalanine {[F₅]f or D-[F₅]Phe},chlorophenylalanine, bromophenylalanine, nitrophenylalanine,D-pyridylalanine, D-homophenylalanine, methyltyrosine, and benzylserine,as well as substitution with D-lysine structure, D-valine structure, ora D-leucine structure. The term “D-leucine structure” is intended toinclude D-leucine, as well as substitution with D-valine, D-isoleucine,or other natural or non-natural amino acids having an aliphatic sidechain, such as D-norleucine, or D-norvaline. The term “D-valinestructure” is intended to include D-valine, as well as substitution withD-leucine or other natural or non-natural amino acid having an aliphaticside chain.

[0042] In other embodiments, the peptidic structure of the modulatorcomprises at least two D-amino acid structures independently selectedfrom the group consisting of a D-leucine structure, a D-phenylalaninestructure, a D-valine structure, a D-alanine structure, a D-tyrosinestructure, a D-iodotyrosine structure, and a D-lysine structure. Inanother embodiment, the peptidic structure is comprised of at leastthree D-amino acid structures independently selected from the groupconsisting of a D-leucine structure, a D-phenylalanine structure and aD-valine structure. In yet another embodiment, the peptidic structure iscomprised of at least three D-amino acid structures independentlyselected from the group consisting of a D-leucine structure, aD-phenylalanine structure, a D-valine structure, a D-alanine structure,a D-tyrosine structure, a D-iodotyrosine structure, and a D-lysinestructure. In yet another embodiment, the peptidic structure comprisesat least four D-amino acid structures independently selected from thegroup consisting of a D-leucine structure, a D-phenylalanine structureand a D-valine structure. In yet another embodiment, the peptidicstructure is comprised of at least four D-amino acid structuresindependently selected from the group consisting of a D-leucinestructure, a D-phenylalanine structure and a D-valine structure. Inpreferred embodiments, the peptidic structure includes at least onephenylalanine analogue which is more stable than phenylalanine and lessprone to, for example, oxidative metabolism.

[0043] In one embodiment, the invention provides a β-amyloid modulatorcompound comprising a formula (I):

[0044] wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each D-amino acidstructures and at least two of Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are,independently, selected from the group consisting of a D-leucinestructure, a D-phenylalanine structure, e.g., D-cyclohexylalanine,D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, and D-homophenylalanine, and a D-valine structure;

[0045] Y, which may or may not be present, is a structure having theformula (Xaa)_(a), wherein Xaa is any D-amino acid structure and a is aninteger from 1 to 15;

[0046] Z, which may or may not be present, is a structure having theformula (Xaa)_(b), wherein Xaa is any D-amino acid structure and b is aninteger from 1 to 15;

[0047] A, which may or may not be present, is a modifying group attacheddirectly or indirectly to the compound; and

[0048] n is an integer from 1 to 15;

[0049] wherein Xaa₁, Xaa₂, Xaa₃, Xaa₄, Y, Z, A and n are selected suchthat the compound binds to natural β-amyloid peptides or modulates theaggregation or inhibits the neurotoxicity of natural β-amyloid peptideswhen contacted with the natural β-amyloid peptides, and is less prone tometabolism, e.g., oxidative metabolism.

[0050] In a subembodiment of this formula, a fifth amino acid residue,Xaa₅, is specified C-terminal to Xaa₄ and Z, which may or may not bepresent, is a structure having the formula (Xaa)_(b), wherein Xaa is anyD-amino acid structure and b is an integer from 1 to 14. Accordingly,the invention provides a β-amyloid modulator compound comprising aformula (II):

[0051] wherein b is an integer from 1 to 14.

[0052] In a preferred embodiment, Xaa₁, Xaa₂, Xaa₃, Xaa₄ of formula (I)are selected based on the sequence of Aβ₁₇₋₂₀, or acceptablesubstitutions thereof Accordingly, in preferred embodiments, Xaa₁ is aD-alanine structure or a D-leucine structure, Xaa₂ is a D-valinestructure or a D-phenylalanine structure, Xaa₃ is a D-phenylalaninestructure, e.g., D-cyclohexylalanine, D-4-fluorophenylalanine(para-fluorophenylalanine), D-pentafluorophenylalanine,chlorophenylalanine, bromophenylalanine, nitrophenylalanine, andD-homophenylalanine, a D-tyrosine structure, a D-iodotyrosine structure,or a D-lysine structure and Xaa₄ is a D-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, and D-homophenylalanine, a D-tyrosine structure, aD-iodotyrosine structure, or a D-lysine structure.

[0053] In another preferred embodiment, Xaa₁, Xaa₂, Xaa₃, Xaa₄ and Xaa₅of formula (II) are selected based on the sequence of Aβ₁₇₋₂₁ oracceptable substitutions thereof. Accordingly, in preferred embodiments,Xaa₁ is a D-alanine structure or a D-leucine structure, Xaa₂ is aD-valine structure, Xaa₃ is a D-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, and D-homophenylalanine, a D-tyrosine structure, aD-iodotyrosine structure, or a D-lysine structure, Xaa₄ is aD-phenylalanine structure, e.g., D-cyclohexylalanine,D4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, D-pyridylalanine, and D-homophenylalanine, aD-tyrosine structure, a D-iodotyrosine structure, or a D-lysinestructure, and Xaa₅ is a D-alanine structure or a D-leucine structure.

[0054] In another preferred embodiment, Xaa₁, Xaa₂, Xaa₃ and Xaa₄ offormula (I) are selected based on the retro-inverso isomer of Aβ₁₇₋₂₀,or acceptable substitutions thereof. Accordingly, in preferredembodiments, Xaa₁ is a D-alanine structure, a D-leucine structure, or aD-phenylalanine structure, e.g., D-cyclohexylalanine,D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, and D-homophenylalanine, a D-tyrosine structure, aD-iodotyrosine structure, a D-leucine structure, a D-valine structure,or a D-lysine structure; Xaa₂ is a D-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, D-pyridylalanine, and D-homophenylalanine, aD-tyrosine structure, a D-iodotyrosine structure, or a D-lysinestructure; Xaa₃ is a D-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, D-pyridylalanine, and D-homophenylalanine, aD-tyrosine structure, a D-iodotyrosine structure, or a D-lysinestructure; and Xaa₄ is a D-valine structure or a D-leucine structure.

[0055] In another preferred embodiment, Xaa₁, Xaa₂, Xaa₃, Xaa₄ and Xaa₅of formula (II) are selected based on the retroinverso isomer ofAβ₁₇₋₂₁, or acceptable substitutions thereof. Accordingly, in preferredembodiments, Xaa₁ is a D-alanine structure, a D-leucine structure or aD-phenylalanine structure, e.g., D-cyclohexylalanine,D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, D-pyridylalanine, and D-homophenylalarune, aD-tyrosine structure, a D-iodotyrosine structure, or a D-lysinestructure; Xaa₂ is a D-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, D-pyridylalanine, and D-homophenylalanine, aD-tyrosine structure, a D-iodotyrosine structure, or a D-lysinestructure; Xaa₃ is a D-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, D-pyridylalanine, and D-homophenylalanine, aD-tyrosine structure, a D-iodotyrosine structure, or a D-lysinestructure; Xaa₄ is a D-valine structure or a D-leucine structure andXaa₅ is a D-leucine structure.

[0056] In another embodiment, the invention provides a β-amyloidmodulator compound comprising a formula (III):

[0057] wherein Xaa₁ and Xaa₂ are each D-amino acid structures and atleast two of Xaa₁ and Xaa₂ are, independently, selected from the groupconsisting of a D-leucine structure, a D-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, and D-homophenylalanine, a D-tyrosine structure, aD-iodotyrosine structure, a D-lysine structure, or a D-valine structure;

[0058] NH—NH is a hydrazine structure;

[0059] Y, which may or may not be present, is a structure having theformula (Xaa)_(a), wherein Xaa is any D-amino acid structure and a is aninteger from 1 to 15;

[0060] Xaa₁′, Xaa₂′, and Xaa₃′ which may or may not be present, are eachD-amino acid or L-amino acid structures and at least two of Xaa₁′,Xaa₂′, and Xaa₃′ are, independently, selected from the group consistingof a D- or L-leucine structure, a D- or L-phenylalanine structure, e.g.,D-cyclohexylalanine, D-4-fluorophenylalanine (para-fluorophenylalanine),D-pentafluorophenylalanine, chlorophenylalanine, bromophenylalanine,nitrophenylalanine, and D-homophenylalanine, a D- or L-tyrosinestructure, a D- or L-iodotyrosine structure, a D- or L-lysine structure,or a D- or L-valine structure;

[0061] Z, which may or may not be present, is a structure having theformula (Xaa)_(b), wherein Xaa is any D-amino acid structure and b is aninteger from 1 to 15;

[0062] A, which may or may not be present, is a modifying group attacheddirectly or indirectly to the compound; and

[0063] n is an integer from 1 to 15;

[0064] wherein Xaa₁, Xaa₂, Xaa₁′, Xaa₂′, Xaa₃′, Y, Z, A and n areselected such that the compound binds to natural β-amyloid peptides ormodulates the aggregation or inhibits the neurotoxicity of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides,and is less prone to metabolism, e.g., oxidative metabolism.

[0065] In the modulators of the invention having the formula (I), (II),or (III) shown above, an optional modifying group (“A”) is attacheddirectly or indirectly to the peptidic structure of the modulator. (Asused herein, the term “modulating group” and “modifying group” are usedinterchangeably to describe a chemical group directly or indirectlyattached to a peptidic structure). For example, a modifying group(s) canbe directly attached by covalent coupling to the peptidic structure or amodifying group(s) can be attached indirectly by a stable non-covalentassociation. In one embodiment of the invention, a modifying group isattached to the amino-terminus of the modulator. Alternatively, inanother embodiment of the invention, a modifying group is attached tothe carboxy-terminus of the modulator. In other embodiments, themodifying group is attached to both the amino and the carboxy-terminusof the modulator. In yet another embodiment, a modulating group(s) isattached to the side chain of at least one amino acid residues of thepeptidic structure of the modulator (e.g., through the epsilon aminogroup of a lysyl residue(s), through the carboxyl group of an asparticacid residue(s) or a glutamic acid residue(s), through a hydroxy groupof a tyrosyl residue(s), a serine residue(s) or a threonine residue(s)or other suitable reactive group on an amino acid side chain).

[0066] If a modifying group(s) is present, the modifying group isselected such that the compound inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.Accordingly, since the β-AP peptide of the compound is modified from itsnatural state, the modifying group “A” as used herein is not intended toinclude hydrogen. In a modulator of the invention, a single modifyinggroup may be attached to the peptidic structure or multiple modifyinggroups may be attached to the peptidic structure. The number ofmodifying groups is selected such that the compound inhibits aggregationof natural β-amyloid peptides when contacted with the natural β-amyloidpeptides. However, n preferably is an integer between 1 and 60, morepreferably between 1 and 30 and even more preferably between 1 and 10 or1 and 5. In a preferred embodiment, A is an amino-terminal modifyinggroup comprising a cyclic, heterocyclic, polycyclic, linear, or branchedalkyl group and n=1. In another preferred embodiment, A iscarboxy-terminally modifying group comprising an amide group, an alkylamide group, an aryl amide group or a hydroxy group, and n=1. Suitablemodifying groups are described further in subsections II and III below.

[0067] In preferred specific embodiments, the invention provides aβ-amyloid modulator compound comprising a peptidic structure selectedfrom the group consisting of (D-Leu-D-Val-D-Phe-D-Cha-D-Leu)(SEQ IDNO:5); (D-Leu-D-Val-D-Cha-D-Phe-D-Leu) (SEQ ID NO:6);(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)(SEQ ID NO:7);(D-Leu-D-Val-D-[p-F]Phe-D-Phe-D-Leu)(SEQ ID NO:8);(D-Leu-D-Val-D-Phe-D-[F-₅]Phe-D-Leu)(SEQ ID NO:9);(D-Leu-D-Val-D-[F₅]Phe-D-Phe-D-Leu)(SEQ ID NO:10);(D-Leu-D-Phe-D-Cha-D-Val-D-Leu)(SEQ ID NO:11);(D-Leu-D-Phe-D-[p-F]Phe-D-Val-D-Leu)(SEQ ID NO:12);D-Leu-D-Phe-D-[F₅]Phe-D-Val-D-Leu)(SEQ ID NO:13);(D-Leu-D-Phe-D-Lys-D-Val-D-Leu)(SEQ ID NO:14);(D-Leu-D-Cha-D-Phe-D-Val-D-Leu)(SEQ ID NO:15);(D-Leu-D-[p-F]Phe-D-Phe-D-Val-D-Leu)(SEQ ID NO:16);(D-Leu-D-[F₅]Phe-D-Phe-D-Val-D-Leu)(SEQ ID NO:17);(D-Leu-D-Lys-D-Phe-D-Val-D-Leu)(SEQ ID NO:18);(D-Leu-D-Cha-D-Cha-D-Val-D-Leu)(SEQ ID NO:19);(D-Leu-D-Val-D-Cha-D-Cha-D-Leu)(SEQ ID NO:20);(D-Leu-D-[p-F]Phe-D-[p-F]Phe-D-Val-D-Leu)(SEQ ID NO:21);(D-Leu-D-Val-D-[p-F]Phe-D-[p-F]Phe-D-Leu)(SEQ ID NO:22);(D-Leu-D-[F₅]Phe-D-[F₅]Phe-D-Val-D-Leu)(SEQ ID NO:23);(D-Leu-D-Val-D-[F₅]Phe-D-[F₅]Phe-D-Leu)(SEQ ID NO:24);(D-Leu-D-Val-D-Phe) (SEQ ID NO:25).

[0068] Any of the aforementioned specific peptidic structures can beamino-terminally and/or carboxy-terminally modified and describedfurther in subsections II and/or III below.

[0069] Particularly preferred modulators of the invention include thefollowing: N,N-dimethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-dimethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-methyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-ethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-isopropyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-isopropylarnide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-dimethylamide;N,N-diethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-diethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-ethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-ethyl-(Gly-D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-methyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-ethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-propyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-diethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ile)-NH₂;H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ala-)-NH₂;H-(D-Ile-D-Ile-D-Phe-D-Phe-D-Ile)-NH₂;H-(D-Nle-D-Val-D-Phe-D-Phe-D-Ala-)-NH₂;H-(D-Nle-D-Val-D-Phe-D-Phe-D-Nle)-NH₂;1-piperidine-acetyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;1-piperidine-acetyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-D-Leu-D-Val-D-Phe-D-Phe-D-Leu-isopropylamide;H-D-Leu-D-Phe-D-Phe-D-Val-D-Leu-isopropylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-methylamide;H-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-methylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-OH;N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-[F₅]Phe-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-Cha-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-[p-F]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-[F₅]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-Lys-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Cha-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[p-F]Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[F₅]Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Lys-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Cha-D-Cha-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[p-F]Phe-D-[p-F]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[F₅]Phe-D-[F₅]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Lys-D-Lys-D-Val-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-[F₅]Phe-D-Leu)-NH₂;H-D-Leu-D-Val-D-Phe-NH-(H-D-Leu-D-Val-D-Phe-)NH;H-D-Leu-D-Val-D-Phe-NH-NH-COCH₃; and H-D-Leu-D-Val-D-Phe-NH-NH₂.

[0070] Even more preferred compounds of the invention include PPI-1319:H-(D-Leu-D-Phe-[p-F]D-Phe-D-Val-D-Leu)-NH₂ and PPI:1019:N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂. (As described above, D-Chastands for D-cyclohexylalanine; [p-F]f or D-[p-F] Phe stands forD-4-fluorophenylalanine (also para-fluorophenylalanine); [F₅]f or D-[F₅]Phe stands for D-pentafluorophenylalanine; and D-Nle stands forD-norleucine).

[0071] The D-amino acid peptidic structures of the modulators of theinvention are further intended to include other peptide modifications,including analogues, derivatives and mimetics, that retain the abilityof the modulator to alter natural β-AP aggregation as described herein.For example, a D-amino acid peptidic structure of a modulator of theinvention may be further modified to increase its stability,bioavailability, and solubility. The terms “analogue”, “derivative” and“mimetic” as used herein are intended to include molecules which mimicthe chemical structure of a D-peptidic structure and retain thefunctional properties of the D-peptidic structure. Approaches todesigning peptide analogs, derivatives and mimetics are known in theart. For example, see Farmer, P. S. in Drug Design (E. J. Ariens, ed.)Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J. B. andAlewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A. andGainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R. M.(1989) Trends Pharmacol. Sci. 10:270. See also Sawyer, T. K. (1995)“Peptidomimetic Design and Chemical Approaches to Peptide Metabolism” inTaylor, M. D. and Amidon, G. L. (eds.) Peptide-Based Drug Design:Controlling Transport and Metabolism, Chapter 17; Smith, A. B. 3rd, etal. (1995) J. Am. Chem. Soc. 117:11113-11123; Smith, A. B. 3rd, et al.(1994) J. Am. Chem. Soc. 116:9947-9962; and Hirschman, R., et al. (1993)J. Am. Chem. Soc. 115:12550-12568.

[0072] As used herein, a “derivative” of a compound X (e.g., a peptideor amino acid) refers to a form of X in which one or more reactiongroups on the compound have been derivatized with a substituent group.Examples of peptide derivatives include peptides in which an amino acidside chain, the peptide backbone, or the amino- or carboxy-terminus hasbeen derivatized (e.g., peptidic compounds with methylated amidelinkages). As used herein an “analogue” of a compound X refers to acompound which retains chemical structures of X necessary for functionalactivity of X yet which also contains certain chemical structures whichdiffer from X. An examples of an analogue of a naturally-occurringpeptide is a peptide which includes one or more non-naturally-occurringamino acids. As used herein, a “mimetic” of a compound X refers to acompound in which chemical structures of X necessary for functionalactivity of X have been replaced with other chemical structures whichmimic the conformation of X. Examples of peptidomimetics includepeptidic compounds in which the peptide backbone is substituted with oneor more benzodiazepine molecules (see e.g., James, G. L. et al. (1993)Science 260:1937-1942).

[0073] Analogues of the modulator compounds of the invention areintended to include compounds in which one or more D-amino acids of thepeptidic structure are substituted with a homologous amino acid suchthat the properties of the original modulator are maintained. Preferablyconservative amino acid substitutions are made at one or more amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), β-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Non-limiting examples ofhomologous substitutions that can be made in the peptidic structures ofthe modulators of the invention include substitution of D-phenylalaninewith D-tyrosine, D-pyridylalanine or D-homophenylalanine, substitutionof D-leucine with D-valine or other natural or non-natural amino acidhaving an aliphatic side chain and/or substitution of D-valine withD-leucine or other natural or non-natural amino acid having an aliphaticside chain.

[0074] The term mimetic, and in particular, peptidomimetic, is intendedto include isosteres. The term “isostere” as used herein is intended toinclude a chemical structure that can be substituted for a secondchemical structure because the steric conformation of the firststructure fits a binding site specific for the second structure. Theterm specifically includes peptide back-bone modifications (i.e., amidebond mimetics) well known to those skilled in the art. Suchmodifications include modifications of the amide nitrogen, the α-carbon,amide carbonyl, complete replacement of the amide bond, extensions,deletions or backbone crosslinks. Several peptide backbone modificationsare known, including ψ[CH₂S], ψ[CH₂NH], ψ[CSNH₂], ψ[NHCO], ψ[COCH₂], andψ[(E) or (Z) CH═CH]. In the nomenclature used above, ψ indicates theabsence of an amide bond. The structure that replaces the amide group isspecified within the brackets.

[0075] Other possible modifications include an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other derivatives of the modulatorcompounds of the invention include C-terminal hydroxymethyl derivatives,O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether),N-terminally modified derivatives including substituted amides such asalkylamides and hydrazides and compounds in which a C-terminalphenylalanine residue is replaced with a phenethylamide analogue (e.g.,Val-Phe-phenethylamide as an analogue of the tripeptide Val-Phe-Phe).

[0076] The modulator compounds of the invention can be incorporated intopharmaceutical compositions (described further in subsection V below)and can be used in detection and treatment methods as described furtherin subsection VI below.

[0077] II. Modifying Groups

[0078] In certain embodiments, the modulator compounds of the inventionare coupled directly or indirectly to at least one modifying group(abbreviated as MG). The term “modifying group” is intended to includestructures that are directly attached to the D-amino acid peptidicstructure (e.g., by covalent coupling), as well as those that areindirectly attached to the peptidic structure (e.g., by a stablenon-covalent association or by covalent coupling to additional aminoacid residues, or mimetics, analogues or derivatives thereof, which mayflank the Aβ-derived D-amino acid peptidic structure). For example, themodifying group can be coupled to the amino-terminus or carboxy-terminusof an Aβ-derived D-amino acid peptidic structure, or to a peptidic orpeptidomimetic region flanking the core domain. Alternatively, themodifying group can be coupled to a side chain of at least one D-aminoacid residue of an Aβ-derived D-amino acid peptidic structure, or to apeptidic or peptidomimetic region flanking the core domain (e.g.,through the epsilon amino group of a lysyl residue(s), through thecarboxyl group of an aspartic acid residue(s) or a glutamic acidresidue(s), through a hydroxy group of a tyrosyl residue(s), a serineresidue(s) or a threonine residue(s) or other suitable reactive group onan amino acid side chain). Modifying groups covalently coupled to theD-amino acid peptidic structure can be attached by means and usingmethods well known in the art for linking chemical structures,including, for example, amide, alkylamino, carbamate, urea or esterbonds.

[0079] The term “modifying group” is intended to include groups that arenot naturally coupled to natural Aβ peptides in their native form.Accordingly, the term “modifying group” is not intended to includehydrogen. The modifying group(s) is selected such that the modulatorcompound alters, and preferably inhibits, aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides orinhibits the neurotoxicity of natural β-amyloid peptides when contactedwith the natural β-amyloid peptides. Although not intending to belimited by mechanism, in embodiments where the modulator comprises amodifying group(s), the modifying group(s) is thought to function as akey pharmacophore that enhances the ability of the modulator to disruptAβ polymerization.

[0080] In a preferred embodiment, the modifying group(s) comprises analkyl group. The term “alkyl” , as used herein, refers to a straight orbranched chain hydrocarbon group having from about 1 to about 10 carbonatoms. Exemplary alkyl groups include methyl, ethyl, dimethyl, diethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,and n-hexyl. An alkyl group may be unsubstituted, or may be substitutedat one or more positions, with, e.g., halogens, alkyls, cycloalkyls,alkenyls, alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros,thiols, amines, imines, amides, phosphonates, phosphines, carbonyls,carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones,aldehydes, esters, —CF₃, —CN, or the like. Preferred alkyls are methyls,ethyls, dimethyls, diethyls, n-propyls, isopropyls.

[0081] In another embodiment, one modifying group, e.g., an alkyl group,is coupled to another modifying group. In yet another embodiment, aD-amino acid in a modulator compound of the invention is modified withtwo modifying groups. Accordingly, preferred modifying groups include a1-piperidine acetyl group.

[0082] In a preferred embodiment, the modifying group(s) comprises acyclic, heterocyclic, polycyclic or branched alkyl group. The term“cyclic group”, as used herein, is intended to include cyclic saturatedor unsaturated (i.e., aromatic) group having from about 3 to 10,preferably about 4 to 8, and more preferably about 5 to 7, carbon atoms.Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cyclooctyl. Cyclic groups may be unsubstituted orsubstituted at one or more ring positions. Thus, a cyclic group may besubstituted with, e.g., halogens, alkyls, cycloalkyls, alkenyls,alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros, thiols amines,imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls,ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones,aldehydes, esters, —CF₃, —CN, or the like.

[0083] The term “heterocyclic group” is intended to include cyclicsaturated or unsaturated (i.e., aromatic) group having from about 3 to10, preferably about 4 to 8, and more preferably about 5 to 7, carbonatoms, wherein the ring structure includes about one to fourheteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane,imidazole, oxazole, piperidine, piperazine, morpholine and pyridine. Theheterocyclic ring can be substituted at one or more positions with suchsubstituents as, for example, halogens, alkyls, cycloalkyls, alkenyls,alkynyls, aryls, other heterocycles, hydroxyl, amino, nitro, thiol,amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls,silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes,esters, —CF₃, —CN, or the like. Heterocycles may also be bridged orfused to other cyclic groups as described below.

[0084] The term “polycyclic group” as used herein is intended to referto two or more saturated or unsaturated (i.e., aromatic) cyclic rings inwhich two or more carbons are common to two adjoining rings, e.g., therings are “fused rings”. Rings that are joined through non-adjacentatoms are termed “bridged” rings. Each of the rings of the polycyclicgroup can be substituted with such substituents as described above, asfor example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls,hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates,phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,selenoethers, ketones, aldehydes, esters, —CF₃, —CN, or the like.

[0085] A preferred polycyclic group is a group containing a cis-decalinstructure. Although not intending to be limited by mechanism, it isthought that the “bent” conformation conferred on a modifying group bythe presence of a cis-decalin structure contributes to the efficacy ofthe modifying group in disrupting Aβ polymerization. Accordingly, otherstructures which mimic the “bent” configuration of the cis-decalinstructure can also be used as modifying groups. An example of acis-decalin containing structure that can be used as a modifying groupis a cholanoyl structure, such as a cholyl group. For example, amodulator compound can be modified at its amino terminus with a cholylgroup by reacting the aggregation core domain with cholic acid, a bileacid. Moreover, a modulator compound can be modified at its carboxyterminus with a cholyl group according to methods known in the art (seee.g., Wess, G. et al. (1993) Tetrahedron Letters, 34:817-822; Wess, G.et al. (1992) Tetrahedron Letters 33:195-198; and Kramer, W. et al.(1992) J. Biol. Chem. 267:18598-18604). Cholyl derivatives and analoguescan also be used as modifying groups. For example, a preferred cholylderivative is Aic (3-(O-aminoethyl-iso)-cholyl), which has a free aminogroup that can be used to further modify the modulator compound (e.g., achelation group for ^(99m)Tc can be introduced through the free aminogroup of Aic). As used herein, the term “cholanoyl structure” isintended to include the cholyl group and derivatives and analoguesthereof, in particular those which retain a four-ring cis-decalinconfiguration. Examples of cholanoyl structures include groups derivedfrom other bile acids, such as deoxycholic acid, lithocholic acid,ursodeoxycholic acid, chenodeoxycholic acid and hyodeoxycholic acid, aswell as other related structures such as cholanic acid, bufalin andresibufogenin (although the latter two compounds are not preferred foruse as a modifying group). Another example of a cis-decalin containingcompound is 5β-cholestan-3α-ol (the cis-decalin isomer of(+)-dihydrocholesterol). For further description of bile acid andsteroid structure and nomenclature, see Nes, W. R. and McKean, M. L.Biochemistry of Steroids and Other Isopentanoids, University Park Press,Baltimore, Md., Chapter 2.

[0086] In addition to cis-decalin containing groups, other polycyclicgroups may be used as modifying groups. For example, modifying groupsderived from steroids or β-lactams may be suitable modifying groups. Inone embodiment, the modifying group is a “biotinyl structure”, whichincludes biotinyl groups and analogues and derivatives thereof (such asa 2-iminobiotinyl group). In another embodiment, the modifying group cancomprise a “fluorescein-containing group”, such as a group derived fromreacting an Aβ-derived peptidic structure with 5-(and6-)-carboxyfluorescein, succinimidyl ester or fluoresceinisothiocyanate. In various other embodiments, the modifying group(s) cancomprise an N-acetylneuraminyl group, a trans-4-cotininecarboxyl group,a 2-imino-1-imidazolidineacetyl group, an (S)-(−)-indoline-2-carboxylgroup, a (−)-menthoxyacetyl group, a 2-norbomaneacetyl group, aγ-oxo-5-acenaphthenebutyryl, a (−)-2-oxo-4-thiazolidinecarboxyl group, atetrahydro-3-furoyl group, a 2-iminobiotinyl group, adiethylenetriaminepentaacetyl group, a 4-morpholinecarbonyl group, a2-thiopheneacetyl group or a 2-thiophenesulfonyl group.

[0087] In addition to the cyclic, heterocyclic and polycyclic groupsdiscussed above, other types of modifying groups can be used in amodulator of the invention. For example, hydrophobic groups and branchedalkyl groups may be suitable modifying groups. Examples include acetylgroups, phenylacetyl groups, phenylacetyl groups, diphenylacetyl groups,triphenylacetyl groups, isobutanoyl groups, 4-methylvaleryl groups,trans-cinnamoyl groups, butanoyl groups and 1-adamantanecarbonyl groups.

[0088] Yet another type of modifying group is a compound that contains anon-natural amino acid that acts as a beta-turn mimetic, such as adibenzofuran-based amino acid described in Tsang, K. Y. et al. (1994) J.Am. Chem. Soc. 116:3988-4005; Diaz, H and Kelly, J. W. (1991)Tetrahedron Letters 41:5725-5728; and Diaz. H et al. (1992) J. Am. Chem.Soc. 114:8316-8318. An example of such a modifying group is apeptide-aminoethyldibenzofuranyl-proprionic acid (Adp) group (e.g.,DDIIL-Adp)(SEQ ID NO: 31). This type of modifying group further cancomprise one or more N-methyl peptide bonds to introduce additionalsteric hindrance to the aggregation of natural β-AP when compounds ofthis type interact with natural β-AP.

[0089] Yet another type of modifying group is an NH—OR group, where theR can be any of the modified or umodified alkyl or cycloalkyl groupsdescribed herein.

[0090] Non-limiting examples of suitable modifying groups, with theircorresponding modifying reagents, are listed below: Modifying GroupModifying Reagent Methyl- Methylamine, Fmoc-D-[Me]-Leu- OH, methylamineand a bromoacetylpeptide Ethyl- Ethylamine, acetaldehyde and sodiumcyanoborohydride, ethylamine and a bromoacetylpeptide Propyl-Propylamine, propionaldehyde and sodium cyanoborohydride, propylamineand a bromoacetylpeptide Isopropyl- Isopropylamine, isopropylamine and abromoacetylpeptide Piperidine- Piperidine and a bromoacetylpeptideAcetyl- Acetic anhydride, acetic acid Dimethyl- Methylamine,formaldehyde and sodium cyanoborohydride Diethyl- Acetaldehyde andsodium cyanoborohydride Cholyl- Cholic acid Lithocholyl- Lithocholicacid Hyodeoxycholyl- Hyodeoxycholic acid Chenodeoxycholyl-Chenodeoxycholic acid Ursodeoxycholyl- Ursodeoxycholic acid3-Hydroxycinnamoyl- 3-Hydroxycinnamic acid 4-Hydroxycinnamoyl-4-Hydroxycinnamic acid 2-Hydroxycinnamoyl- 2-Hydroxycinnamic acid3-Hydroxy-4-methoxycinnamoyl- 3-Hydroxy-4-methoxycinnamic acid4-Hydroxy-3-methoxycinnamoyl- 4-Hydroxy-3-methoxycinnamic acid2-Carboxycinnamoyl- 2-Carboxycinnamic acid 3-Formylbenzoyl3-Carboxybenzaldehyde 4-Formylbenzoyl 4-Carboxybenzaldehyde3,4,-Dihydroxyhydrocinnamoyl- 3,4,-Dihydroxyhydrocinnamic acid3,7-Dihydroxy-2-napthoyl- 3,7-Dihydroxy-2-naphthoic acid4-Formylcinnamoyl- 4-Formylcinnamic acid 2-Formylphenoxyacetyl-2-Formylphenoxyacetic acid 8-Formyl-1-napthoyl 1,8-napthaldehydic acid4-(hydroxymethyl)benzoyl- 4-(hydroxymethyl)benzoic acid4-Hydroxyphenylacetyl- 4-Hydroxyphenylacetic acid 3-Hydroxybenzoyl-3-Hydroxybenzoic acid 4-Hydroxybenzoyl- 4-Hydroxybenzoic acid5-Hydantoinacetyl- 5-Hydantoinacetic acid L-Hydroorotyl- L-Hydrooroticacid 4-Methylvaleryl- 4-Methylvaleric acid 2,4-Dihydroxybenzoyl-2,4-Dihydroxybenzoic acid 3,4-Dihydroxycinnamoyl- 3,4-Dihydroxycinnamicacid 3,5-Dihydroxy-2-naphthoyl- 3,5-Dihydroxy-2-naphthoic acid3-Benzoylpropanoyl- 3-Benzoylpropanoic acid trans-Cinnamoyl-trans-Cinnamic acid Phenylacetyl- Phenylacetic acid Diphenylacetyl-Diphenylacetic acid Triphenylacetyl- Triphenylacetic acid2-Hydroxyphenylacetyl- 2-Hydroxyphenylacetic acid 3-Hydroxyphenylacetyl-3-Hydroxyphenylacetic acid 4-Hydroxyphenylacetyl- 4-Hydroxyphenylaceticacid (±)-Mandelyl- (±)-Mandelic acid (±)-2,4-Dihydroxy-3,3-(±)-Pantolactone dimethylbutanoyl Butanoyl- Butanoic anhydrideIsobutanoyl- Isobutanoic anhydride Hexanoyl- Hexanoic anhydridePropionyl- Propionic anhydride 3-Hydroxybutyroyl β-Butyrolactone4-Hydroxybutyroyl γ-Butyrolactone 3-Hydroxypropionoyl β-Propiolactone2,4-Dihydroxybutyroyl α-Hydroxy-β-Butyrolactone 1-Adamantanecarbonyl-1-Adamantanecarbonic acid Glycolyl- Glycolic acidDL-3-(4-hydroxyphenyl)lactyl- DL-3-(4-hydroxyphenyl)lactic acid3-(2-Hydroxyphenyl)propionyl- 3-(2-Hydroxyphenyl)propionic acid4-(2-Hydroxyphenyl)propionyl- 4-(2-Hydroxyphenyl)propionic acidD-3-Phenyllactyl- D-3-Phenyllactic acid Hydrocinnamoyl- Hydrocinnamicacid 3-(4-Hydroxyphenyl)propionyl- 3-(4-Hydroxyphenyl)propionic acidL-3-Phenyllactyl- L-3-Phenyllactic acid 4-methylvaleryl 4-methylvalericacid 3-pyridylacetyl 3-pyridylacetic acid 4-pyridylacetyl4-pyridylacetic acid Isonicotinoyl 4-quinolinecarboxyl4-quinolinecarboxylic acid 1-isoquinolinecarboxyl1-isoquinolinecarboxylic acid 3-isoquinolinecarboxyl3-isoquinolinecarboxylic acid

[0091] Preferred modifying groups include methyl-containing groups,ethyl-containing groups, propyl-containing groups, andpiperidine-containing groups, e.g., a 1-piperidine-acetyl group.

[0092] III. Additional Chemical Modifications of AD Modulators

[0093] A β-amyloid modulator compound of the invention can be furthermodified to alter the specific properties of the compound whileretaining the ability of the compound to alter Aβ aggregation andinhibit Aβ neurotoxicity. For example, in one embodiment, the compoundis further modified to alter a pharmacokinetic property of the compound,such as in vivo stability or half-life. In another embodiment, thecompound is further modified to label the compound with a detectablesubstance. In yet another embodiment, the compound is further modifiedto couple the compound to an additional therapeutic moiety.Schematically, a modulator of the invention comprising a D-amino acid Aβaggregation core domain coupled directly or indirectly to at least onemodifying group can be illustrated as MG-ACD, whereas this compoundwhich has been further modified to alter the properties of the modulatorcan be illustrated as MG-ACD-CM, wherein CM represents an additionalchemical modification.

[0094] To further chemically modify the compound, such as to alter thepharmacokinetic properties of the compound, reactive groups can bederivatized. For example, when the modifying group is attached to theamino-terminal end of the aggregation core domain, the carboxy-terminalend of the compound can be further modified. Preferred C-terminalmodifications include those which reduce the ability of the compound toact as a substrate for carboxypeptidases. Examples of preferredC-terminal modifiers include an amide group (i.e., a peptide amide), analkyl or aryl amide group (e.g., an ethylamide group or a phenethylamidegroup) a hydroxy group (i.e., a peptide alcohol) and various non-naturalamino acids, such as D-amino acids and β-alanine. Alternatively, whenthe modifying group is attached to the carboxy-terminal end of theaggregation core domain, the amino-terminal end of the compound can befurther modified, for example, to reduce the ability of the compound toact as a substrate for aminopeptidases.

[0095] A modulator compound can be further modified to label thecompound by reacting the compound with a detectable substance. Suitabledetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,^(99m)Tc, ³⁵S or ³H. In a preferred embodiment, a modulator compound isradioactively labeled with ¹⁴C, either by incorporation of ¹⁴C into themodifying group or one or more amino acid structures in the modulatorcompound. Labeled modulator compounds can be used to assess the in vivopharmacokinetics of the compounds, as well as to detect Aβ aggregation,for example for diagnostic purposes. Aβ aggregation can be detectedusing a labeled modulator compound either in vivo or in an in vitrosample derived from a subject.

[0096] Preferably, for use as an in vivo diagnostic agent, a modulatorcompound of the invention is labeled with radioactive technetium oriodine. Accordingly, in one embodiment, the invention provides amodulator compound labeled with technetium, preferably ^(99m)Tc. Methodsfor labeling peptide compounds with technetium are known in the art (seee.g., U.S. Pat. Nos. 5,443,815, 5,225,180 and 5,405,597, all by Dean etal.; Stepniak-Biniakiewicz, D., et al. (1992) J. Med Chem. 35:274-279;Fritzberg, A. R., et al. (1988) Proc. Natl. Acad. Sci. USA 85:4025-4029;Baidoo, K. E., et al. (1990) Cancer Res. Suppl. 50:799s-803s; and Regan,L. and Smith, C. K. (1995) Science 270:980-982). A modifying group canbe chosen that provides a site at which a chelation group for ^(99m)Tccan be introduced, such as the Aic derivative of cholic acid, which hasa free amino group. In another embodiment, the invention provides amodulator compound labeled with radioactive iodine. For example, aphenylalanine residue within the Aβ sequence (such as Phe₁₉ or Phe₂₀)can be substituted with radioactive iodotyrosyl. Any of the variousisotopes of radioactive iodine can be incorporated to-create adiagnostic agent. Preferably, ¹²³I (half-life=13.2 hours) is used forwhole body scintigraphy, ¹²⁴I(half life=4 days) is used for positronemission tomography (PET), ¹²⁵I (half life=60 days) is used formetabolic turnover studies and ¹³¹I(half life=8 days) is used for wholebody counting and delayed low resolution imaging studies.

[0097] Furthermore, an additional modification of a modulator compoundof the invention can serve to confer an additional therapeutic propertyon the compound. That is, the additional chemical modification cancomprise an additional functional moiety. For example, a functionalmoiety which serves to break down or dissolve amyloid plaques can becoupled to the modulator compound. In this form, the MG-ACD portion ofthe modulator serves to target the compound to Aβ peptides and disruptthe polymerization of the Aβ peptides, whereas the additional functionalmoiety serves to break down or dissolve amyloid plaques after thecompound has been targeted to these sites.

[0098] In an alternative chemical modification, a β-amyloid compound ofthe invention is prepared in a “prodrug” form, wherein the compounditself does not modulate AP aggregation, but rather is capable of beingtransformed, upon metabolism in vivo, into a β-amyloid modulatorcompound as defined herein. For example, in this type of compound, themodulating group can be present in a prodrug form that is capable ofbeing converted upon metabolism into the form of an active modulatinggroup. Such a prodrug form of a modifying group is referred to herein asa “secondary modifying group. ” A variety of strategies are known in theart for preparing peptide prodrugs that limit metabolism in order tooptimize delivery of the active form of the peptide-based drug (seee.g., Moss, J. (1995) in Peptide-Based Drug Design: ControllingTransport and Metabolism, Taylor, M. D. and Amidon, G. L. (eds), Chapter18. Additionally strategies have been specifically tailored to achievingCNS delivery based on “sequential metabolism” (see e.g., Bodor, N., etal. (1992) Science 257:1698-1700; Prokai, L., et al. (1994) J. Am. Chem.Soc. 116:2643-2644; Bodor, N. and Prokai, L. (1995) in Peptide-BasedDrug Design: Controlling Transport and Metabolism, Taylor, M. D. andAmidon, G. L. (eds), Chapter 14. In one embodiment of a prodrug form ofa modulator of the invention, the modifying group comprises an alkylester to facilitate blood-brain barrier permeability.

[0099] Modulator compounds of the invention can be prepared by standardtechniques known in the art. The peptide component of a modulator can besynthesized using standard techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant, G. A (ed.). Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). Automated peptide synthesizers arecommercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Additionally, one or more modulating groupscan be attached to the Aβ-derived peptidic component (e.g., an Aβaggregation core domain) by standard methods, for example using methodsfor reaction through an amino group (e.g., the alpha-amino group at theamino-terminus of a peptide), a carboxyl group (e.g., at the carboxyterminus of a peptide), a hydroxyl group (e.g., on a tyrosine, serine orthreonine residue) or other suitable reactive group on an amino acidside chain (see e.g., Greene, T. W and Wuts, P. G. M. Protective Groupsin Organic Synthesis, John Wiley and Sons, Inc., New York (1991).Exemplary syntheses of D-amino acid β amyloid modulator are describedfurther in Example 1.

[0100] IV. Screening Assays

[0101] Another aspect of the invention pertains to a method forselecting a modulator of β-amyloid aggregation. In the method, a testcompound is contacted with natural β amyloid peptides, the aggregationof the natural β-AP is measured and a modulator is selected based on theability of the test compound to alter the aggregation of the naturalβ-AP (e.g., inhibit or promote aggregation). In a preferred embodiment,the test compound is contacted with a molar excess amount of the naturalβ-AP. The amount and/or rate of natural β-AP aggregation in the presenceof the test compound can be determined by a suitable assay indicative ofβ-AP aggregation, as described herein (see e.g., Example 2).

[0102] In a preferred assay, the natural β-AP is dissolved in solutionin the presence of the test compound and aggregation of the natural β-APis assessed in a nucleation assay (see Example 2) by assessing theturbidity of the solution over time, as measured by the apparentabsorbance of the solution at 405 nm (described further in Example 2;see also Jarrett et al. (1993) Biochemistry 32:4693-4697). In theabsence of a β-amyloid modulator, the A_(405nm) of the solutiontypically stays relatively constant during a lag time in which the β-APremains in solution, but then the A_(405nm) of the solution rapidlyincreases as the β-AP aggregates and comes out of solution, ultimatelyreaching a plateau level (i.e., the A_(405nm) of the solution exhibitssigmoidal kinetics over time). In contrast, in the presence of a testcompound that inhibits β-AP aggregation, the A_(405nm) of the solutionis reduced compared to when the modulator is absent. Thus, in thepresence of the inhibitory modulator, the solution may exhibit anincreased lag time, a decreased slope of aggregation and/or a lowerplateau level compared to when the modulator is absent. This method forselecting a modulator of β-amyloid polymerization can similarly be usedto select modulators that promote β-AP aggregation. Thus, in thepresence of a modulator that promotes β-AP aggregation, the A_(405nm) ofthe solution is increased compared to when the modulator is absent(e.g., the solution may exhibit an decreased lag time, increase slope ofaggregation and/or a higher plateau level compared to when the modulatoris absent).

[0103] Another assay suitable for use in the screening method of theinvention, a seeded extension assay, is also described further inExample 2. In this assay, β-AP monomer and an aggregated β-AP “seed” arecombined, in the presence and absence of a test compound, and the amountof β-fibril formation is assayed based on enhanced emission of the dyeThioflavine T when contacted with β-AP fibrils. Moreover, β-APaggregation can be assessed by electron microscopy (EM) of the β-APpreparation in the presence or absence of the modulator. For example, βamyloid fibril formation, which is detectable by EM, is reduced in thepresence of a modulator that inhibits β-AP aggregation (i.e., there is areduced amount or number of β-fibrils in the presence of the modulator),whereas β fibril formation is increased in the presence of a modulatorthat promotes β-AP aggregation (i.e., there is an increased amount ornumber of β-fibrils in the presence of the modulator).

[0104] Another preferred assay for use in the screening method of theinvention to select suitable modulators is the neurotoxicity assaydescribed in Example 3. Compounds are selected which inhibit theformation of neurotoxic Aβ aggregates and/or which inhibit theneurotoxicity of preformed Aβ fibrils. This neurotoxicity assay isconsidered to be predictive of neurotoxicity in vivo. Accordingly,inhibitory activity of a modulator compound in the in vitroneurotoxicity assay is predictive of similar inhibitory activity of thecompound for neurotoxicity in vivo.

[0105] V. Pharmaceutical Compositions

[0106] Another aspect of the invention pertains to pharmaceuticalcompositions of the β-amyloid modulator compounds of the invention. Inone embodiment, the composition includes a β amyloid modulator compoundin a therapeutically or prophylactically effective amount sufficient toalter, and preferably inhibit, aggregation of natural β-amyloidpeptides, and a pharmaceutically acceptable carrier. In anotherembodiment, the composition includes a β amyloid modulator compound in atherapeutically or prophylactically effective amount sufficient toinhibit the neurotoxicity of natural β-amyloid peptides, and apharmaceutically acceptable carrier. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result, such asreduction or reversal or β-amyloid deposition and/or reduction orreversal of Aβ neurotoxicity. A therapeutically effective amount ofmodulator may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the modulator toelicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the modulator are outweighed by the therapeutically beneficialeffects. The potential neurotoxicity of the modulators of the inventioncan be assayed using the cell-based assay described in Example 6 and atherapeutically effective modulator can be selected which does notexhibit significant neurotoxicity. In a preferred embodiment, atherapeutically effective amount of a modulator is sufficient to alter,and preferably inhibit, aggregation of a molar excess amount of naturalβ-amyloid peptides. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result, such as preventing orinhibiting the rate of β-amyloid deposition and/or Aβ neurotoxicity in asubject predisposed to β-amyloid deposition. A prophylacticallyeffective amount can be determined as described above for thetherapeutically effective amount. Typically, since a prophylactic doseis used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

[0107] One factor that may be considered when determining atherapeutically or prophylactically effective amount of a β amyloidmodulator is the concentration of natural β-AP in a biologicalcompartment of a subject, such as in the cerebrospinal fluid (CSF) ofthe subject. The concentration of natural β-AP in the CSF has beenestimated at 3 nM (Schwartzman, (1994) Proc. Natl. Acad. Sci. USA91:8368-8372). A non-limiting range for a therapeutically orprophylactically effective amounts of a β amyloid modulator is 0.01nM-10 μM. It is to be noted that dosage values may vary with theseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

[0108] The amount of active compound in the composition may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, each of which may affect the amount of natural β-AP inthe individual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

[0109] As used herein “pharmaceutically acceptable carrier” includes anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. In one embodiment, the carrier issuitable for parenteral administration. Preferably, the carrier issuitable for administration into the central nervous system (e.g.,intraspinally or intracerebrally). Alternatively, the carrier can besuitable for intravenous, intraperitoneal or intramuscularadministration. In another embodiment, the carrier is suitable for oraladministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

[0110] Therapeutic compositions typically must be sterile and stableunder the conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, monostearate salts and gelatin. Moreover, themodulators can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Theactive compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations arepatented or generally known to those skilled in the art.

[0111] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., β-amyloid modulator) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

[0112] A modulator compound of the invention can be formulated with oneor more additional compounds that enhance the solubility of themodulator compound. Preferred compounds to be added to formulations toenhance the solubility of the modulators are cyclodextrin derivatives,preferably hydroxypropyl-γ-cyclodextrin. Drug delivery vehiclescontaining a cyclodextrin derivative for delivery of peptides to thecentral nervous system are described in Bodor, N., et al. (1992) Science257:1698-1700. For the β-amyloid modulators described herein, inclusionin the formulation of hydroxypropyl-γ-cyclodextrin at a concentration50-200 mM increases the aqueous solubility of the compounds. In additionto increased solubility, inclusion of a cyclodextrin derivative in theformulation may have other beneficial effects, since β-cyclodextrinitself has been reported to interact with the Aβ peptide and inhibitfibril formation in vitro (Carnilleri, P., et al. (1994) FEBS Letters341:256-258. Accordingly, use of a modulator compound of the inventionin combination with a cyclodextrin derivative may result in greaterinhibition of Aβ aggregation than use of the modulator alone. Chemicalmodifications of cyclodextrins are known in the art (Hanessian, S., etal. (1995) J. Org. Chem. 60:4786-4797). In addition to use as anadditive in a pharmaceutical composition containing a modulator of theinvention, cyclodextrin derivatives may also be useful as modifyinggroups and, accordingly, may also be covalently coupled to an Aβ peptidecompound to form a modulator compound of the invention.

[0113] Another preferred formulation for the modulator compounds toenhance brain uptake comprises the detergent Tween-80, polyethyleneglycol (PEG) and ethanol in a saline solution. A non-limiting example ofsuch a preferred formulation is 0.16% Tween-80, 1.3% PEG-3000 and 2%ethanol in saline.

[0114] In another embodiment, a pharmaceutical composition comprising amodulator of the invention is formulated such that the modulator istransported across the blood-brain barrier (BBB). Various strategiesknown in the art for increasing transport across the BBB can be adaptedto the modulators of the invention to thereby enhance transport of themodulators across the BBB (for reviews of such strategies, see e.g.,Pardridge, W. M. (1994) Trends in Biotechnol. 12:239-245; Van Bree, J.B. et al. (1993) Pharm. World Sci. 15:2-9; and Pardridge, W. M. et al.(1992) Pharmacol. Toxicol. 71:3-10). In one approach, the modulator ischemically modified to form a prodrug with enhanced transmembranetransport. Suitable chemical modifications include covalent linking of afatty acid to the modulator through an amide or ester linkage (see e.g.,U.S. Pat. No. 4,933,324 and PCT Publication WO 89/07938, both byShashoua; U.S. Pat. No. 5,284,876 by Hesse et al.; Toth, I. et al.(1994) J. Drug Target. 2:217-239; and Shashoua, V. E. et al. (1984) J.Med. Chem. 27:659-664) and glycating the modulator (see e.g., U.S. Pat.No. 5,260,308 by Poduslo et al.). Also, N-acylamino acid derivatives maybe used in a modulator to form a “lipidic” prodrug (see e.g., U.S. Pat.No. 5,112,863 by Hashimoto et al.).

[0115] In another approach for enhancing transport across the BBB, apeptidic or peptidomimetic modulator is conjugated to a second peptideor protein, thereby forming a chimeric protein, wherein the secondpeptide or protein undergoes absorptive-mediated or receptor-mediatedtranscytosis through the BBB. Accordingly, by coupling the modulator tothis second peptide or protein, the chimeric protein is transportedacross the BBB. The second peptide or protein can be a ligand for abrain capillary endothelial cell receptor ligand. For example, apreferred ligand is a monoclonal antibody that specifically binds to thetransferrin receptor on brain capillary endothelial cells (see e.g.,U.S. Pat. No. 5,182,107 and 5,154,924 and PCT Publications WO 93/10819and WO 95/02421, all by Friden et al. ). Other suitable peptides orproteins that can mediate transport across the BBB include histones (seee.g., U.S. Pat. No. 4,902,505 by Pardridge and Schimmel) and ligandssuch as biotin, folate, niacin, pantothenic acid, riboflavin, thiamin,pryridoxal and ascorbic acid (see e.g., U.S. Pat. No. 5,416,016 and5,108,921, both by Heinstein). Additionally, the glucose transporterGLUT-1 has been reported to transport glycopeptides(L-serinyl-β-D-glucoside analogues of [Met5]enkephalin) across the BBB(Polt, R. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7114-1778).Accordingly, a modulator compound can be coupled to such a glycopeptideto target the modulator to the GLUT-1glucose transporter. For example, amodulator compound which is modified at its amino terminus with themodifying group Aic (3-(O-aminoethyl-iso)-cholyl, a derivative of cholicacid having a free amino group) can be coupled to a glycopeptide throughthe amino group of Aic by standard methods. Chimeric proteins can beformed by recombinant DNA methods (e.g., by formation of a chimeric geneencoding a fusion protein) or by chemical crosslinking of the modulatorto the second peptide or protein to form a chimeric protein. Numerouschemical crosslinking agents are known in the (e.g., commerciallyavailable from Pierce, Rockford Ill.). A crosslinking agent can bechosen which allows for high yield coupling of the modulator to thesecond peptide or protein and for subsequent cleavage of the linker torelease bioactive modulator. For example, a biotin-avidin-based linkersystem may be used.

[0116] In yet another approach for enhancing transport across the BBB,the modulator is encapsulated in a carrier vector which mediatestransport across the BBB. For example, the modulator can be encapsulatedin a liposome, such as a positively charged unilamellar liposome (seee.g., PCT Publications WO 88/07851 and WO 88/07852, both by Faden) or inpolymeric microspheres (see e.g., U.S. Pat. No. 5,413,797 by Khan etal., U.S. Pat. No. 5,271,961 by Mathiowitz et al. and U.S. Pat. No.5,019,400 by Gombotz et al.). Moreover, the carrier vector can bemodified to target it for transport across the BBB. For example, thecarrier vector (e.g., liposome) can be covalently modified with amolecule which is actively transported across the BBB or with a ligandfor brain endothelial cell receptors, such as a monoclonal antibody thatspecifically binds to transferrin receptors (see e.g., PCT PublicationsWO 91/04014 by Collins et al. and WO 94/02178 by Greig et al.).

[0117] In still another approach to enhancing transport of the modulatoracross the BBB, the modulator is coadministered with another agent whichfunctions to permeabilize the BBB. Examples of such BBB “permeabilizers”include bradykinin and bradykinin agonists (see e.g., U.S. Pat. No.5,112,596 by Malfroy-Camine) and peptidic compounds disclosed in U.S.Pat. No. 5,268,164 by Kozarich et al.

[0118] Assays that measure the in vitro stability of the modulatorcompounds in cerebrospinal fluid (CSF) and the degree of brain uptake ofthe modulator compounds in animal models can be used as predictors of invivo efficacy of the compounds. Suitable assays for measuring CSFstability and brain uptake are described in Examples 7 and 8,respectively.

[0119] A modulator compound of the invention can be formulated into apharmaceutical composition wherein the modulator is the only activecompound or, alternatively, the pharmaceutical composition can containadditional active compounds. For example, two or more modulatorcompounds may be used in combination. Moreover, a modulator compound ofthe invention can be combined with one or more other agents that haveanti-amyloidogenic properties. For example, a modulator compound can becombined with the non-specific cholinesterase inhibitor tacrine(COGNEX®, Parke-Davis).

[0120] In another embodiment, a pharmaceutical composition of theinvention is provided as a packaged formulation. The packagedformulation may include a pharmaceutical composition of the invention ina container and printed instructions for administration of thecomposition-for treating a subject having a disorder associated withβ-amyloidosis, e.g. Alzheimer's disease.

[0121] VI. Methods of Using Aβ Modulators

[0122] Another aspect of the invention pertains to methods for alteringthe aggregation or inhibiting the neurotoxicity of natural β-amyloidpeptides. In the methods of the invention, natural β amyloid peptidesare contacted with a β amyloid modulator such that the aggregation ofthe natural β amyloid peptides is altered or the neurotoxicity of thenatural β amyloid peptides is inhibited. In a preferred embodiment, themodulator inhibits aggregation of the natural β amyloid peptides. Inanother embodiment, the modulator promotes aggregation of the natural βamyloid peptides. Preferably, aggregation of a molar excess amount ofβ-AP, relative to the amount of modulator, is altered upon contact withthe modulator.

[0123] In the method of the invention, natural β amyloid peptides can becontacted with a modulator either in vitro or in vivo. Thus, the term“contacted with” is intended to encompass both incubation of a modulatorwith a natural β-AP preparation in vitro and delivery of the modulatorto a site in vivo where natural β-AP is present. Since the modulatorcompound interacts with natural β-AP, the modulator compounds can beused to detect natural β-AP, either in vitro or in vivo. Accordingly,one use of the modulator compounds of the invention is as diagnosticagents to detect the presence of natural β-AP, either in a biologicalsample or in vivo in a subject. Furthermore, detection of natural β-APutilizing a modulator compound of the invention further can be used todiagnose amyloidosis in a subject. Additionally, since the modulatorcompounds of the invention disrupt β-AP aggregation and inhibit β-APneurotoxicity, the modulator compounds also are useful in the treatmentof disorders associated with β-amyloidosis, either prophylactically ortherapeutically. Accordingly, another use of the modulator compounds ofthe invention is as therapeutic agents to alter aggregation and/orneurotoxicity of natural β-AP.

[0124] In one embodiment, a modulator compound of the invention is usedin vitro, for example to detect and quantitate natural β-AP in sample(e.g., a sample of biological fluid). To aid in detection, the modulatorcompound can be modified with a detectable substance. The source ofnatural β-AP used in the method can be, for example, a sample ofcerebrospinal fluid (e.g., from an AD patient, an adult susceptible toAD due to family history, or a normal adult). The natural β-AP sample iscontacted with a modulator of the invention and aggregation of the β-APis measured, such as by the assays described in Example 2. The degree ofaggregation of the β-AP sample can then be compared to that of a controlsample(s) of a known concentration of β-AP, similarly contacted with themodulator and the results can be used as an indication of whether asubject is susceptible to or has a disorder associated withβ-amyloidosis. Moreover, β-AP can be detected by detecting a modulatinggroup incorporated into the modulator. For example, modulatorsincorporating a biotin compound as described herein (e.g., anamino-terminally biotinylated β-AP peptide) can be detected using astreptavidin or avidin probe which is labeled with a detectablesubstance (e.g., an enzyme, such as peroxidase).

[0125] In another embodiment, a modulator compound of the invention isused in vivo to detect, and, if desired, quantitate, natural β-APdeposition in a subject, for example to aid in the diagnosis of βamyloidosis in the subject. To aid in detection, the modulator compoundcan be modified with a detectable substance, preferably ^(99m)Tc orradioactive iodine (described further above), which can be detected invivo in a subject. The labeled β-amyloid modulator compound isadministered to the subject and, after sufficient time to allowaccumulation of the modulator at sites of amyloid deposition, thelabeled modulator compound is detected by standard imaging techniques.The radioactive signal generated by the labeled compound can be directlydetected (e.g., whole body counting), or alternatively, the radioactivesignal can be converted into an image on an autoradiograph or on acomputer screen to allow for imaging of amyloid deposits in the subject.Methods for imaging amyloidosis using radiolabeled proteins are known inthe art. For example, serum amyloid β component (SAP), radiolabeled witheither ¹²³I or ^(99m)Tc, has been used to image systemic amyloidosis(see e.g., Hawkins, P. N. and Pepys, M. B. (1995) Eur. J. Nucl. Med.22:595-599). Of the various isotypes of radioactive iodine, preferably¹²³I (half-life=13.2 hours) is used for whole body scintigraphy, ¹²⁴I(half life=4 days) is used for positron emission tomography (PET), ¹²⁵I(half life=60 days) is used for metabolic turnover studies and ¹³¹I(half life=8 days) is used for whole body counting and delayed lowresolution imaging studies. Analogous to studies using radiolabeled SAP,a labeled modulator compound of the invention can be delivered to asubject by an appropriate route (e.g., intravenously, intraspinally,intracerebrally) in a single bolus, for example containing 100 μg oflabeled compound carrying approximately 180 MBq of radioactivity.

[0126] The invention provides a method for detecting the presence orabsence of natural β-amyloid peptides in a biological sample, comprisingcontacting a biological sample with a compound of the invention anddetecting the compound bound to natural β-amyloid peptides to therebydetect the presence or absence of natural β-amyloid peptides in thebiological sample. In one embodiment, the β-amyloid modulator compoundand the biological sample are contacted in vitro. In another embodiment,the β-amyloid modulator compound is contacted with the biological sampleby administering the β-amyloid modulator compound to a subject. For invivo administration, preferably the compound is labeled with radioactivetechnetium or radioactive iodine.

[0127] The invention also provides a method for detecting naturalβ-amyloid peptides to facilitate diagnosis of a β-amyloidogenic disease,comprising contacting a biological sample with the compound of theinvention and detecting the compound bound to natural β-amyloid peptidesto facilitate diagnosis of a β-amyloidogenic disease. In one embodiment,the β-amyloid modulator compound and the biological sample are contactedin vitro. In another embodiment, the β-amyloid modulator compound iscontacted with the biological sample by administering the β-amyloidmodulator compound to a subject. For in vivo administration, preferablythe compound is labeled with radioactive technetium or radioactiveiodine. Preferably, use of the method facilitates diagnosis ofAlzheimer's disease.

[0128] In another embodiment, the invention provides a method foraltering natural β-AP aggregation or inhibiting β-AP neurotoxicity,which can be used prophylactically or therapeutically in the treatmentor prevention of disorders associated with β amyloidosis, e.g.,Alzheimer's Disease. Modulator compounds of the invention can reduce thetoxicity of natural β-AP aggregates to cultured neuronal cells.Moreover, the modulators also have the ability to reduce theneurotoxicity of preformed Aβ fibrils. Accordingly, the modulatorcompounds of the invention can be used to inhibit or prevent theformation of neurotoxic Aβ fibrils in subjects (e.g., prophylacticallyin a subject predisposed to β-amyloid deposition) and can be used toreverse β-amyloidosis therapeutically in subjects already exhibitingβ-amyloid deposition.

[0129] A modulator of the invention is contacted with natural β amyloidpeptides present in a subject (e.g., in the cerebrospinal fluid orcerebrum of the subject) to thereby alter the aggregation of the naturalβ-AP and/or inhibit the neurotoxicity of the natural β-APs. A modulatorcompound alone can be administered to the subject, or alternatively, themodulator compound can be administered in combination with othertherapeutically active agents (e.g., as discussed above in subsectionIV). When combination therapy is employed, the therapeutic agents can becoadministered in a single pharmaceutical composition, coadministered inseparate pharmaceutical compositions or administered sequentially.

[0130] The modulator may be administered to a subject by any suitableroute effective for inhibiting natural β-AP aggregation in the subject,although in a particularly preferred embodiment, the modulator isadministered parenterally, most preferably to the central nervous systemof the subject. Possible routes of CNS administration includeintraspinal administration and intracerebral administration (e.g.,intracerebrovascular administration). Alternatively, the compound can beadministered, for example, orally, intraperitoneally, intravenously orintramuscularly. For non-CNS administration routes, the compound can beadministered in a formulation which allows for transport across the BBB.Certain modulators may be transported across the BBB without anyadditional further modification whereas others may need furthermodification as described above in subsection IV.

[0131] Suitable modes and devices for delivery of therapeutic compoundsto the CNS of a subject are known in the art, including cerebrovascularreservoirs (e.g., Ommaya or Rikker reservoirs; see e.g., Raney, J. P. etal. (1988) J. Neurosci. Nurs. 20:23-29; Sundaresan, N. et al. (1989)Oncology 3:15-22), catheters for intrathecal delivery (e.g.,Port-a-Cath, Y-catheters and the like; see e.g., Plummer, J. L. (1991)Pain 44:215-220; Yaksh, T. L. et al. (1986) Pharmacol. Biochem. Behav.25:483-485), injectable intrathecal reservoirs (e.g., Spinalgesic; seee.g., Brazenor, G. A. (1987) Neurosurgery 21:484-491), implantableinfusion pump systems (e.g., Infusaid; see e.g., Zierski, J. et al.(1988) Acta Neurochem. Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am.Osteopath. Assoc. 94:487-493) and osmotic pumps (sold by AlzaCorporation). A particularly preferred mode of administration is via animplantable, externally programmable infusion pump. Suitable infusionpump systems and reservoir systems are also described in U.S. Pat. No.5,368,562 by Blomquist and U.S. Pat. No. 4,731,058 by Doan, developed byPharmacia Deltec Inc.

[0132] The method of the invention for altering β-AP aggregation in vivo, and in particular for inhibiting β-AP aggregation, can be usedtherapeutically in diseases associated with abnormal β amyloidaggregation and deposition to thereby slow the rate of β amyloiddeposition and/or lessen the degree of β amyloid deposition, therebyameliorating the course of the disease. In a preferred embodiment, themethod is used to treat Alzheimer's disease (e.g., sporadic or familialAD, including both individuals exhibiting symptoms of AD and individualssusceptible to familial AD). The method can also be usedprophylactically or therapeutically to treat other clinical occurrencesof β amyloid deposition, such as in Down's syndrome individuals and inpatients with hereditary cerebral hemorrhage with amyloidosis-Dutch-type(HCHWA-D). While inhibition of PMAP aggregation is a preferredtherapeutic method, modulators that promote β-AP aggregation may also beuseful therapeutically by allowing for the sequestration of β-AP atsites that do not lead to neurological impairment.

[0133] Additionally, abnormal accumulation of β-amyloid precursorprotein in muscle fibers has been implicated in the pathology ofsporadic inclusion body myositis (IBM) (Askana, V. et al. (1996) Proc.Natl. Acad. Sci. USA 93:1314-1319; Askanas, V. et al. (1995) CurrentOpinion in Rheumatology 7:486-496). Accordingly, the modulators of theinvention can be used prophylactically or therapeutically in thetreatment of disorders in which β-AP, or APP, is abnormally deposited atnon-neurological locations, such as treatment of IBM by delivery of themodulators to muscle fibers.

[0134] This invention is further illustrated by the following exampleswhich should not be construed as limiting. A modulator's ability toalter the aggregation of natural β-amyloid peptide and/or inhibit theneurotoxicity of natural β-amyloid peptide in the assays described beloware predictive of the modulator's ability to perform the same functionin vivo.

[0135] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures, are hereby incorporated byreference.

EXAMPLE 1 Preparation of β-amyloid Modulator Compounds ComprisingD-Amino Acids

[0136] β-amyloid modulators comprising D-amino acids can be prepared bysolid-phase peptide synthesis, for example using anN^(α)-9-fluorenylmethyloxycarbonyl (FMOC)-based protection strategy asfollows. Starting with 2.5 mmoles of FMOC-D-Val-Wang resin, sequentialadditions of each amino acid are performed using a four-fold excess ofprotected amino acids, 1-hydroxybenzotriazole (HOBt) and diisopropylcarbodiimide (DIC). Recouplings are performed when necessary asdetermined by ninhydrin testing of the resin after coupling. Eachsynthesis cycle is minimally described by a three minute deprotection(25% piperidine/N-methyl-pyrrolidone (NMP)), a 15 minute deprotection,five one minute NMP washes, a 60 minute coupling cycle, five NMP washesand a ninhydrin test. For N-terminal modification, an N-terminalmodifying reagent is substituted for an FMOC-D-amino acid and coupled toa 700 mg portion of the fully assembled peptide-resin by the aboveprotocol. The peptide is removed from the resin by treatment withtrifluoroacetic acid (TFA)(82.5%), water (5%), thioanisole (5%), phenol(5%), ethanedithiol (2.5%) for two hours followed by precipitation ofthe peptide in cold ether. The solid is pelleted by centriftigation(2400 rpm×10 min.), and the ether decanted. The solid is resuspended inether, pelleted and decanted a second time. The solid is dissolved in10% acetic acid and lyophilized to dryness. For preparative purificationand subsequent analytical characterization, 60 mg of the solid isdissolved in 25% acetonitrile (ACN)/0.1% TFA and applied to a C18reversed phase high performance liquid chromatography (HPLC) column.

[0137] Alternatively, β-amyloid modulators comprising D-amino acids canbe prepared on a Rainin PS3 peptide synthesizer using an automatedprotocol established by the manufacturer for a 0.25 mmole scalesynthesis. Couplings are performed using2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium-hexafluoro-phosphate(HBTU)/FMOC-D-amino acid in four fold excess in 0.4 M N-methylmorpholine(NMM)/dimethylformamide (DMF) for 60 minutes. In between couplings, theFMOC group is removed by reaction with 20% piperidine/DMF for 20minutes. The peptide is removed from the resin by treatment with 95%TFA/water for one hour and precipitated with ether. The pellet isresuspended in 40% acetonitrile/water and lyophilized. When necessary,the material was purified by preparative HPLC using 15%-50% acetonitrileover 60 minutes on a Vydac C18 column (21×250 mm).

[0138] Various N-terminally modified β-amyloid modulator compounds canbe synthesized using standard methods. Fully-protected resin-boundpeptides are prepared as described above on an appropriate resin toeventually afford carboxyl terminal peptide acids. Small portions ofeach peptide resin (e.g., 13-20 μmoles) are aliquoted into separatereaction vessels. The N-terminal FMOC protecting group of each sample isremoved in the standard manner with 20% piperidine in NMM followed byextensive washing with DMF. The unprotected N-terminal a-amino group ofeach peptide-resin sample can be modified using one of the followingmethods:

[0139] Method A, coupling of modifying reagents containing freecarboxylic acid groups: The modifying reagent (five equivalents) ispredissolved in NMP, DMSO or a mixture of these two solvents. HOBT andDIC (five equivalents of each reagent) are added to the dissolvedmodifier and the resulting solution is added to one equivalent offree-amino peptide-resin. Coupling is allowed to proceed overnight,followed by washing. If a ninhydrin test on a small sample ofpeptide-resin shows that coupling is not complete, the coupling isrepeated using 1-hydroxy-7-azabenzotriazole (HOAt) in place of HOBt.

[0140] Method B, coupling of modifying reagents obtained in preactivatedforms: The modifying reagent (five equivalents) is predissolved in NMP,DMSO or a mixture of these two solvents and added to one equivalent ofpeptide-resin. Diisopropylethylamine (DIEA; six equivalents) is added tothe suspension of activated modifier and peptide-resin. Coupling isallowed to proceed overnight, followed by washing. If a ninhydrin teston a small sample of peptide-resin shows that coupling is not complete,the coupling is repeated.

[0141] After the second coupling (if required) the N-terminally modifiedpeptide-resins are dried at reduced pressure and cleaved from the resinwith removal of side-chain protecting groups as described above.Analytical reversed-phase HPLC is used to confirm that a major productis present in the resulting crude peptides, which are purified usingMillipore Sep-Pak cartridges or preparative reverse-phase HPLC. Massspectrometry or high-field nuclear magnetic resonance spectrometry isused to confirm the presence of the desired compound in the product.

[0142] Method C, preparation of N-terminal-alkyl substituted peptidesusing bromoacetyl peptide intermediates: A resin-bound peptide can becoupled to bromoacetic acid (12 equivalents) with1,3-diisopropylcarbodiimide (DIC)(13 equivalents) in DMF. The resultingbromoacetyl substituted peptide can be modified upon reaction withprimary or secondary arnines including, methylamine, ethylamine,propylamine, isopropylamine and piperidine. The reaction is performed in60% DMSO/DMF and is typically complete after 24 hours.

[0143] Method D, preparation of N-terminal-alkyl substituted peptidesvia reductive alkylation: After the peptide is dissolved (or partiallydissolved) in water containing 0-10% methanol, it is reacted with analdehyde (5-8 equivalents) and sodiumcyanoborohydride (10-16equivalents). The number of equivalents can be adjusted for the. type ofaldehyde and the degree of substitution desired. The pH of the resultingsolution is adjusted to 2 with 1 M HCl and maintained at 2 for one hour.The reaction is monitored by hplc and is usually completed with twohours. The reaction mix is concentrated at room temperature and hplcpurified.

[0144] Method E, C-terminal modification: The peptide was synthesized on2-chlorotrityl resin using standard Fmoc chemistry however the finalD-amino acid group coupled was Boc protected. The peptide was removedfrom the resin with 8/1/1 dichloromethane (DCM)/aceticacid/trifluoroethanol and the mixture concentrated. The peptide residuewas dissolved in 20% acetonitrile, frozen and lyophilyzed overnight. Thecrude BOC protected peptide acid was coupled under basic conditions(pH=11, adjusted with DIEA) to an amine with one equivalent each of1-hydroxy-7-azobenzotriazole(HOAt) and DIC. The reaction was completedafter stirring overnight and the peptide precipitated with water. TheBOC group was cleaved upon reaction with 25% TFA in DCM for one hour andthe peptide HPLC purified.

EXAMPLE 2 β-Amyloid Aggregation Assays

[0145] The ability of β-amyloid modulator compounds to modulate (e.g.,inhibit or promote) the aggregation of natural β-AP when combined withthe natural β-AP can be examined in one or both of the aggregationassays described below. Natural β-AP (β-AP₁₋₄₀) for use in theaggregation assays is commercially available from Bachem (Torrance,Calif.).

[0146] A. Nucleation Assay

[0147] The nucleation assay is employed to determine the ability of testcompounds to alter (e.g. inhibit) the early events in formation of β-APfibers from monomeric β-AP. Characteristic of a nucleated polymerizationmechanism, a lag time is observed prior to nucleation, after which thepeptide rapidly forms fibers as reflected in a linear rise in turbidity.The time delay before polymerization of β-AP monomer can be quantifiedas well as the extent of formation of insoluble fiber by lightscattering (turbidity). Polymerization reaches equilibrium when themaximum turbidity reaches a plateau. The turbidity of a solution ofnatural β-AP in the absence or presence of various concentrations of aβ-amyloid modulator compound is determined by measuring the apparentabsorbance of the solution at 405 nm (A_(405 nm) ) over time. Thethreshold of sensitivity for the measurement of turbidity is in therange of 15-20 μM β-AP. A decrease in turbidity over time in thepresence of the modulator, as compared to the turbidity in the absenceof the modulator, indicates that the modulator inhibits formation ofβ-AP fibers from monomeric β-AP. This assay can be performed usingstirring or shaking to accelerate polymerization, thereby increasing thespeed of the assay. Moreover the assay can be adapted to a 96-well plateformat to screen multiple compounds.

[0148] To perform the nucleation assay, first Aβ₁₋₄₀ peptide isdissolved in HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol; Aldrich 10,522-8)at a concentration of 2 mg peptide/ml and incubated at room temperaturefor 30 min. HFIP-solubilized peptide is sonicated in a waterbathsonicator for 5 min at highest setting, then evaporated to dryness undera stream of argon. The peptide film is resuspended in anhydrousdimethylsulfoxide (DMSO) at a concentration of 6.9 mg/ml (25×concentration), sonicated for 5 min as before, then filtered through a0.2 micron nylon syringe filter (VWR cat. No. 28196-050). Test compoundsare dissolved in DMSO at a 100× concentration. Four volumes of 25×Aβ₁₋₄₀ peptide in DMSO are combined with one volume of test compound inDMSO in a glass vial, and mixed to produce a 1:1 molar ratio of Aβpeptide to test compound. For different molar ratios, test compounds arediluted with DMSO prior to addition to Aβ₁₋₄₀, in order to keep thefinal DMSO and Aβ₁₋₄₀ concentrations constant. Control samples do notcontain the test compound. Ten microliters of the mixture is then addedto the bottom of a well of a Corning Costar ultra low binding 96-wellplate (Coming Costar, Cambridge Mass.; cat. No. 2500). Ninetymicroliters of water is added to the well, the plate is shaken on arotary shaken at a constant speed at room temperature for 30 seconds, anadditional 100 μl of 2× PTL buffer (20 mM NaH₂PO₄, 300 mM NaCl, pH 7.4)is added to the well, the plate is reshaken for 30 seconds and abaseline (t=0) turbidity reading is taken by measuring the apparentabsorbance at 405 nm using a Bio-Rad Model 450 Microplate Reader. Theplate is then returned to the shaker and shaken continuously for 5hours. Turbidity readings are taken at 15 minute intervals.

[0149] β-amyloid aggregation in the absence of any modulators results inenhanced turbidity of the natural β-AP solution (i.e., an increase inthe apparent absorbance at 405 nm over time). Accordingly, a solutionincluding an effective inhibitory modulator compound exhibits reducedturbidity as compared to the control sample without the modulatorcompound (ie., less apparent absorbance at 405 nm over time as comparedto the control sample).

[0150] Alternative to use of turbidity to quantitate β-amyloidaggregation, fluorescence of thioflavin T (Th-T) also can be used toquantitate β-amyloid aggregation in the nucleation assay (use of Th-Tfluorescence for quantitating β-amyloid aggregation is described furtherbelow for the seeded extension assay).

[0151] B. Fibril Binding Assay

[0152] The following materials are needed for the Fibril binding assay:Millipore multifilter apparatus; 12×75 glass tubes; GF/F 25 mm glassfilters; PBS/0.1% tween 20 at 4° C. (PBST); Aβ fibrils; radioactivecompound; nonradioactive compound; Eppendorf repeat pipettor with tips;labels; forceps; and vacuum.

[0153] In this assay, each sample is run in triplicate. The “aged” Aβfibril is first prepared approximately 8 days in advance by aging 1 mlaliquots of a 200 μM Aβ1-40 peptide solution in 4%DMSO/PBS for 8 days at37° C. with rocking. Such “aged” Aβ peptide can be tested directly oncells or frozen at −80° C.

[0154] The 200 μM Aβ fibril is diluted in PBST to yield a 4 μM solution(320 μl in 16 ml PBST). 100 μL aliquots of this solution are added pertube with the repeat pipettor.

[0155] The β-amyloid modulator compounds of the invention are preparedat 2 μM-200 fM dilutions as follows:

[0156] Dilute a 5 mM stock 1:3 in DMSO to yield a 1.6667 stock (200 μlin 400 μl DMSO).

[0157] Dilute a 1.667 mM stock 1:3 in DMSO to yield a 0.5556 stock (200μl in 400 μl DMSO).

[0158] Dilute a 555.556 μM stock 1:3 in DMSO to yield a 185.19 stock(200 μl in 400 μl DMSO).

[0159] 5 Dilute a 185.185 μM stock 1:3 in DMSO to yield a 61.728 stock(200 μl in 400 μl DMSO).

[0160] Dilute a 61.728 μM stock 1:3 in DMSO to yield a 20.576 stock (200μl in 400 μl DMSO).

[0161] Dilute a 20.576 μM stock 1:3 in DMSO to yield a 6.8587 stock (200μl in 400 μl DMSO).

[0162] Dilute a 6.859 μM stock 1:3 in DMSO to yield a 2.2862 stock (200μl in 400 μl DMSO).

[0163] Dilute a 2.286 μM stock 1:3 in DMSO to yield a 0.7621 stock (200μl in 400 μl DMSO).

[0164] 10 Dilute a 762.079 nM stock 1:3 in DMSO to yield a 254.03 stock(200 μl in 400 μl DMSO).

[0165] Dilute a 254.026 nM stock 1:3 in DMSO to yield a 84.675 stock(200 μl in 400 μl DMSO).

[0166] Dilute a 84.675 nM stock 1:3 in DMSO to yield a 28.225 stock (200μl in 400 μl DMSO).

[0167] Dilute a 28.225 nM stock 1:3 in DMSO to yield a 9.4084 stock (200μl in 400 μl DMSO).

[0168] Dilute a 9.408 nM stock 1:3 in DMSO to yield a 3.1361 stock (200μl in 400 μl DMSO).

[0169] 15 Dilute a 3.136 nM stock 1:3 in DMSO to yield a 1.0454 stock(200 μl in 400 μl DMSO).

[0170] Dilute a 1.045 nM stock 1:3 in DMSO to yield a 0.3485 stock (200μl in 400 μl DMSO).

[0171] Dilute a 348.459 pM stock 1:3 in DMSO to yield a 116.15 stock(200 μl in 400 μl DMSO).

[0172] Dilute a 116.153 pM stock 1:3 in DMSO to yield a 38.718 stock(200 μl in 400 μl DMSO).

[0173] Dilute 185.185 μM stock 1:25 in PBST to yield 7.4074 (50 μL in1.2 mL PBST)

[0174] 20 Dilute 61.728 μM stock 1:25 in PBST to yield 2.4691 (50 μL in1.2 mL PBST)

[0175] Dilute 20.576 μM stock 1:25 in PBST to yield 0.823 (50 μL in 1.2mL PBST)

[0176] Dilute 6.859 μM stock 1:25 in PBST to yield 0.2743 (50 μL in 1.2mL PBST)

[0177] Dilute 2.286 μM stock 1:25 in PBST to yield 0.0914 (50 μL in 1.2mL PBST)

[0178] Dilute 762.079 nM stock 1:25 in PBST to yield 30.483 (50 μL in1.2 mL PBST)

[0179] 25 Dilute 254.026 nM stock 1:25 in PBST to yield 10.161 (50 μL in1.2 mL PBST)

[0180] Dilute 84.675 nM stock 1:25 in PBST to yield 3.387 (50 μL in 1.2mL PBST)

[0181] Dilute 28.225 nM stock 1:25 in PBST to yield 1.129 (50 μL in 1.2mL PBST)

[0182] Dilute 9.408 nM stock 1:25 in PBST to yield 0.3763 (50 μL in 1.2mL PBST)

[0183] Dilute 3.136 nM stock 1:25 in PBST to yield 0.1254 (50 μL in 1.2mL PBST)

[0184] 30 Dilute 1.045 nM stock 1:25 in PBST to yield 0.0418 (50 μL in1.2 mL PBST)

[0185] Dilute 348.459 pM stock 1:25 in PBST to yield 13.938 (50 μL in1.2 mL PBST)

[0186] Dilute 116.153 pM stock 1:25 in PBST to yield 4.6461 (50 μL in1.2 mL PBST)

[0187] The β-amyloid modulator compound (200μL) is then added to theappropriate tube containing the Aβ fibril.

[0188] The radioactively labeled β-amyloid modulator compound isprepared using standard radioactive safety protocols by making adilution into a PBS/0.1% tween-20 solution such that there is a finalconcentration of 20,000 dpm per 100 μL. 100 μl aliqouots of theradioactively labeled p-amyloid modulator compound are added per tubeusing the repeat pipettor. The samples are covered with parafilm andincubated at 37° C. inside plastic radioactivity bags overnight.

[0189] To filter the samples, the filters are pre-wetted in a smallvolume of PBST. Two Millipore multifiltration apparati are set with GF/Ffilters in each filtration slot following the instructions from themanufacturer. The samples are removed from the 37° C. incubator and eachsample is filtered using a small volume (˜5 ml) of cold PBST buffer. Thesample tube is then washed with two additional 5 mL volumes of cold PBSTbuffer. The vacuum is allowed to pull to a semi dry filter forapproximately 2 minutes after adding the last sample and the filter istransferred to a labelled tube for iodination counting. One minutecounts are recorded, the data is plotted, and the Prism program(GraphPAD) is used to analyze the graph, according to the manufacturer'sinstrutions.

[0190] C. Seeded Extension Assay

[0191] The seeded extension assay can be employed to measure the rate ofAβ fiber formed in a solution of Aβ monomer following addition ofpolymeric Aβ fiber “seed”. The ability of test compounds to preventfurther deposition of monomeric Aβ to previously deposited amyloid isdetermined using a direct indicator of β-sheet formation usingfluorescence. In contrast with the nucleation assay, the addition ofseed provides immediate nucleation and continued growth of preformedfibrils without the need for continuous mixing, and thus results in theabsence of a lag time before polymerization starts. Since this assayuses static polymerization conditions, the activity of positivecompounds in the nucleation assay can be confirmed in this second assayunder different conditions and with an additional probe of amyloidstructure.

[0192] In the seeded extension assay, monomeric Aβ₁₋₄₀ is incubated inthe presence of a “seed” nucleus (approximately ten mole percent of Aβthat has been previously allowed to polymerize under controlled staticconditions). Samples of the solution are then diluted in thioflavin T(Th-T). The polymer-specific association of Th-T with Aβ produces afluorescent complex that allows the measurement of the extent of fibrilformation (Levine, H. (1993) Protein Science 2:404-410). In particular,association of Th-T with aggregated β-AP, but not monomeric or looselyassociated β-AP, gives rise to a new excitation (ex) maximum at 450 nmand an enhanced emission (em) at 482 nm, compared to the 385 nm (ex) and445 nm (em) for the free dye. Small aliquots of the polymerizationmixture contain sufficient fibril to be mixed with Th-T to allow themonitoring of the reaction mixture by repeated sampling. A linear growthcurve is observed in the presence of excess monomer. The formation ofthioflavin T responsive β-sheet fibrils parallels the increase inturbidity observed using the nucleation assay.

[0193] A solution of Aβ monomer for use in the seeded extension assay isprepared by dissolving an appropriate quantity of Aβ₁₋₄₀ peptide in{fraction (1/25)} volume of dimethysulfoxide (DMSO), followed by waterto ½ volume and ½ volume 2×PBS (10×PBS: NaCl 137 mM, KCI 2.7 mMNa₂HPO₄·7H₂O 4.3 mM, KH₂PO₄ 1.4 mM pH 7.2) to a final concentration of200 μM. To prepare the stock seed, 1 ml of the Aβ monomer preparation,is incubated for approximately 8 days at 37° C. and sheared sequentiallythrough an 18, 23, 26 and 30 gauge needle 25, 25, 50, and 100 timesrespectively. 2 μl samples of the sheared material is taken forfluorescence measurements after every 50 passes through the 30 gaugeneedle until the fluorescence units (FU) plateau (approx. 100-150×).Test compounds are prepared by dissolving an appropriate amount of testcompound in 1×PBS to a final concentration of 1 mM (10×stock). Ifinsoluble, the compound is dissolved in {fraction (1/10)} volume of DMSOand diluted in 1×PBS to 1 mM. A further {fraction (1/10)} dilution isalso prepared to test each candidate at both 100 μM and 10μM.

[0194] To perform the seeded extension assay, each sample is set up with50 μl of 200 μM monomer, 125 FU sheared seed (a variable quantitydependent on the batch of seed, routinely 3-6 μl) and 10 μl of10×modulator solution. The sample volume is then adjusted to a finalvolume of 100 μl with 1×PBS. Two concentrations of each modulatortypically are tested: 100 μM and 10 μM, equivalent to a 1:1 and a 1:10molar ratio of monomer to modulator. The controls include an unseededreaction to confirm that the fresh monomer contains no seed, and aseeded reaction in the absence of any modulators, as a reference tocompare against candidate modulators. The assay is incubated at 37° C.for 6 h, taking 2 μl samples hourly for fluorescence measurements. Tomeasure fluorescence, a 2 μl sample of Aβ is added to 400 μl ofThioflavin-T solution (50 mM Potassium Phosphate 10 mM Thioflavin-T pH7.5). The samples are vortexed and the fluorescence is read in a 0.5 mlmicro quartz cuvette at EX 450 nm and EM482 nm (Hitachi 4500Fluorimeter).

[0195] β-amyloid aggregation results in enhanced emission ofThioflavin-T. Accordingly, samples including an effective inhibitorymodulator compound exhibit reduced emission as compared to controlsamples without the modulator compound.

EXAMPLE 3 Analysis of β-Amyloid Modulator Compounds

[0196] In this example, β-amyloid modulator compounds described hereinwere prepared and tested for their ability to inhibit aggregation ofnatural β-amyloid peptide using aggregations assays as described inExample 2. The results from a first series of experiments, aresummarized below in Tables I, II, and III. TABLE I Nucleation assay Δlag 5 2.5 1.25 Fibril binding Kd's PPI# μM μM μM cmpd ref cmpd ref Kd 803 <1 <1 <1  913 1 1 1  968 >5 >5 2  969 >5 >5 3 1.13 × 10⁻⁹  PPI-558 3.7 × 10⁻⁹  970 >5 >5 1  992 3 1 1 2.43 × 10⁻⁹  PPI-558 3.70 × 10⁻⁹ 993 1 1 1 1005 3 3 1 1006 1 1 1 *1007  4 4 3 8.64 × 10⁻¹⁰ PPI-558 1.69× 10⁻⁹ #1007  1.5 1.5 1.5 6.27 × 10⁻¹⁰ PPI-558 2.75 × 10⁻⁹ 1008 1.75 ×10⁻⁹  PPI-558 1.00 × 10⁻⁹ #1013  2 >3 2 2.47 × 10⁻¹⁰ PPI-558 1.69 × 10⁻⁹1017 3.89 × 10⁻¹⁰ PPI-558 2.42 × 10⁻⁹ 1018 7.01 × 10⁻¹⁰ PPI-558 2.42 ×10⁻⁹ 1020 6.01 × 10⁻¹⁰ PPI-558 2.42 × 10⁻⁹ 1022 1.50 × 10⁻¹⁰ PPI-5581.00 × 10⁻⁹ 1025 4.30 × 10⁻¹⁰ PPI-558 1.00 × 10⁻⁹ 1028 4.90 × 10⁻¹⁰PPI-558 1.00 × 10⁻⁹ 1038 6.52 × 10⁻¹⁰ PPI-558 3.76 × 10⁻⁹ 1039 2.44 ×10⁻¹⁰ PPI-558 3.76 × 10⁻⁹ 1040 4.08 × 10⁻¹⁰ PPI-558  2.4 × 10⁻⁹ 10411.61 × 10⁻⁹  PPI-558  2.4 × 10⁻⁹ 1042 2.34 × 10⁻¹⁰ PPI-558  2.4 × 10⁻⁹1088 3.40 × 10⁻⁹  PPI-558 1.93 × 10⁻⁹ 1089  5.7 × 10⁻¹⁰ PPI-558  3.3 ×10⁻⁹ 1093 1.02 × 10⁻⁹  PPI-558 1.93 × 10⁻⁹ 1094 3.7 × 10⁻⁹ PPI-558  3.5× 10⁻⁹ 1179 6.04 × 10⁻¹⁰ PPI-558 1.93 × 10⁻⁹ 1180  3.3 × 10⁻¹⁰ PPI-558 3.5 × 10⁻⁹ 1261 1.12 × 10⁻⁸  PPI-558 3.34 × 10⁻⁹

[0197] TABLE II Nucleation assay data 3 1 0.3 Fibril binding Kd's PPI#μM μM μM cmpd ref cmpd ref Kd *1019  >2.5 >2.5 2.0 4.11 × 10⁻¹⁰ PPI-558 1.69 × 10⁻⁹ 1019 5.34 × 10⁻¹⁰ PPI-558  1.93 × 10⁻⁹ 1301 1.1 × 10⁻⁹PPI-1318  1.4 × 10⁻⁹ 1302  2.2 × 10⁻¹⁰ PPI-1318  1.4 × 10⁻⁹ 1303 1.1 ×10⁻⁹ PPI-1318  1.4 × 10⁻⁹ 1318 >5 2 1  7.7 × 10⁻¹¹ PPI-558   2.3 × 10⁻⁹1318 1.4 × 10⁻⁹ 1318  6.2 × 10⁻¹¹ 1319 >5 >5 1 1320 >5 3 1 1.4 × 10⁻⁹PPI-1318  6.2 × 10⁻¹¹ 1321 <1 <1 <1 1322 1.2 × 10⁻⁹ PPI-1318  6.2 ×10⁻¹¹ 1323 1324 1325 1.4 × 10⁻⁹ PPI-1318  1.4 × 10⁻⁹ 1326  5.6 × 10⁻¹⁰PPI-1318  6.2 × 10⁻¹¹ 1327  8.2 × 10⁻¹⁰ PPI-1318  1.4 × 10⁻⁹ 1328 2.4 ×10⁻⁹ PPI-1318  6.2 × 10⁻¹¹ 1329 *1125  >2.5 >2.5 2.0 1.27 × 10⁻⁹ PPI-558  2.08 × 10⁻⁹ 1125 1.34 × 10⁻⁹  PPI-558  5.05 × 10⁻⁹ 1133 3.18 ×10⁻⁷  PPI-558  2.08 × 10⁻⁹ 1155 1.24 × 10⁻⁷  PPI-558  2.08 × 10⁻⁹

[0198] The modulator compounds were evaluated in nucleation assaysutilizing 5 μM Aβ₁₋₄₀ and either 5 μM, 2.5 μM, 1.25 μM, 3 μM, 1 μM, or0.3 μM test compound. The change in lag time (ΔLag) is presented as theratio of the lag time observed in the presence of the test compound (ateither 5 μM, 2.5 μM, 1.25 μM, 3 μM, 1 μM, or 0.3 μM) to the lag time ofthe control. TABLE III Fibril binding Kd's PPI# STRUCTURE dpm PPI-504TFA • H-(lv-[3-I]y-fa)-NH₂ PPI-1181 TFA • H-(lvffl)-NH—Et PPI-1465 TFA •H-lvffl-NH—CH₂—CH₂—NH₂ 3.6 × 10⁻⁹ PPI-1603 TFA • H-(GGClvffl)-N₂PPI-1604 TFA • H-(GGClvfyl)-NH₂ PPI-1605 TFA • H-(GGClvf-[3-I]y-l)-NH₂PPI-1619 2TFA • H-LVF-NH—NH-FVL-H 3.5 × 10⁻⁸ (an analog of 1125)PPI-1621 2TFA • H-LVF-NH—NH-fvl-H 8.7 × 10⁻⁹ (an analog of 1125)PPI-1635 TFA • H-lff-(nvl)-l-NH₂ 1.4 × 10⁻⁹ PPI-1636 TFA •H-lf-[pF]f-(nvl)-l-NH₂ 1.5 × 10⁻⁹ PPI-1637 TFA •H-l-[pF]f-[pF]f-(nvl)-l-NH₂ 1.8 × 10⁻⁹ PPI-1782 TFA • Me-lvyfl-NH₂PPI-1783 TFA • H-(lvyfl)-NH₂ PPI-1784 TFA • Me-(lv-[p-F]f-fl)-NH₂ 2.5 ×10⁻⁹ PPI-1785 TFA • H-(lv-[p-F]f-fl)-NH₂ 2.8 × 10⁻⁹ PPI-1786 TFA •H-(lvf-[p-F]f-l)-NH₂ PPI-1787 TFA • Me-lvff-[nvl])-NH₂ 5.8 × 10⁻⁹PPI-1788 TFA • Me-(lvff-[nle])-NH₂  (˜4 × 10⁻⁹) 3-point assay PPI-1799TFA • Me-(lvffl)-OH PPI-1800 TFA • Me-(lvffl)-NH—OH  (˜4 × 10⁻⁹) 3-pointassay PPI-1805 TFA • H-(lv-[p-F]f-f-(nvl))-NH₂ PPI-1806 TFA •Me-(l-v-[p-F]f-f-(nvl))-NH₂ PPI 1807 TFA • H-((nvl)-v-[p-F]f-f-nvl)-NH₂PPI-1818 TFA • H-(l-(nvl)-[p-F]f-f-(nvl)-NH₂ PPI 1819 TFA •H-((nvl)-(nvl)-[p-F]f-f-(nvl))- NH₂ PPI 1820 TFA •Me-(l-(nvl)-[p-F]f-f-(nvl))- NH₂ PPI 1827 TFA • H-(lvff-(nvl))-NH₂ PPI1828 Ac-(lvffl)-NH₂ PPI 1829 Ac-(lvffl)-OH PPI 1830 TFA •H-(lv-[3-I]y-fl)-NH₂

[0199] PPI-1801 is the acetyl amide analog of H-LPFFD-OH that has beenreported in the literature. This compound was prepared and tested foractivity for comparison purposes, The results indicated that thiscompound binds poorly to fibrils in the assay used herein.

[0200] In contrast, the results shown in Tables I, II, and III and FIG.2, demonstrated that β-amyloid modulators of the invention are effectiveinhibitors of Aβ aggregation.

EXAMPLE 6 Neurotoxicity Assay

[0201] The neurotoxicity of natural β-amyloid peptide aggregates, ineither the presence or absence of a β-amyloid modulator, can be testedin a cell-based assay using either a rat or human neuronally-derivedcell line (PC-12 cells or NT-2 cells, respectively) and the viabilityindicator 3, (4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide(MTT). (See e.g., Shearman, M. S. et al. (1994) Proc. Natl. Acad. Sci.USA 91:1470-1474; Hansen, M. B. et al. (1989) J. Immun. Methods119:203-210 for a description of similar cell-based viability assays).PC-12 is a rat adrenal pheochromocytoma cell line and is available fromthe American Type Culture Collection, Rockville, Md. (ATCC CRL 1721).MTT (commercially available from Sigma Chemical Co.) is a chromogenicsubstrate that is converted from yellow to blue in viable cells, whichcan be detected spectrophotometrically.

[0202] To test the neurotoxicity of natural β-amyloid peptides, stocksolutions of fresh Aβ monomers and aged Aβ aggregates are firstprepared. Aβ₁₋₄₀ in 100% DMSO is prepared from lyophilized powder andimmediately diluted in one half the final volume in H₂O and then onehalf the final volume in 2×PBS so that a final concentration of 200 μMpeptide, 4% DMSO is achieved. Peptide prepared in this way and testedimmediately on cells is referred to as “fresh” Aβ monomer. To prepare“aged” Aβ aggregates, peptide solution is placed in a 1.5 ml Eppendorftube and incubated at 37° C. for eight days to allow fibrils to form.Such “aged” Aβ peptide can be tested directly on cells or frozen at −80°C. The neurotoxicity of fresh monomers and aged aggregates are testedusing PC12 and NT2 cells. PC12 cells are routinely cultured inDulbecco's modified Eagle's medium (DMEM) containing 10% horse serum, 5%fetal calf serum, 4mM glutamine, and 1% gentamycin. NT2 cells areroutinely cultured in OPTI-MEM medium (GIBCO BRL CAT. #31985)supplemented with 10% fetal calf serum, 2 mM glutamine and 1%gentamycin. Cells are plated at 10-15,000 cells per well in 90 μl offresh medium in a 96-well tissue culture plate 3-4 hours prior totreatment. The fresh or aged Aβ peptide solutions (10 μL) are thendiluted 1:10 directly into tissue culture medium so that the finalconcentration is in the range of 1-10 μM peptide. Cells are incubated inthe presence of peptide without a change in media for 48 hours at 37° C.For the final three hours of exposure of the cells to the β-APpreparation, MTT is added to the media to a final concentration of 1mg/ml and incubation is continued at 37° C. Following the two hourincubation with MTT, the media is removed and the cells are lysed in 100μL isopropanol/0.4N HCI with agitation. An equal volume of PBS is addedto each well and the plates are agitated for an additional 10 minutes.Absorbance of each well at 570 nm is measured using a microtiter platereader to quantitate viable cell.

[0203] Using this assay, the neurotoxicity of aged (5 day or 8 day)Aβ₁₋₄₀ aggregates alone, but not fresh Aβ₁₋₄₀ monomers alone, wasconfirmed. Experiments demonstrated that incubating the neuronal cellswith increasing amounts of fresh Aβ₁₋₄₀ monomers was not significantlytoxic to the cells whereas incubating the cells with increasing amountsof 5 day or 8 day Aβ₁₋₄₀ aggregates led to increasing amount ofneurotoxicity. The EC₅₀ for toxicity of aged Aβ₁₋₄₀ aggregates was 1-2μM for both the PC 12 cells and the NT2 cells.

[0204] To determine the effect of a β-amyloid modulator compound on theneurotoxicity of Aβ₁₋₄₀ aggregates, a modulator compound is preincubatedwith Aβ₁₋₄₀ monomers under standard nucleation assay conditions asdescribed in Example 2 and at particular time intervals post-incubation,aliquots of the β-AP/modulator solution are removed and 1) the turbidityof the solution is assessed as a measure of aggregation and 2) thesolution is applied to cultured neuronal cells for 48 hours at whichtime cell viability is assessed using MTT to determine the neurotoxicityof the solution. Additionally, the ability of β-amyloid modulatorcompounds to reduce the neurotoxicity of preformed Aβ₁₋₄₀ aggregates canbe assayed. In these experiments, Aβ₁₋₄₀ aggregates are preformed byincubation of the monomers in the absence of any modulators. Themodulator compound is then incubated with the preformed Aβ₁₋₄₀aggregates for 24 hours at 37° C., after which time the β-AP/modulatorsolution is collected and its neurotoxicity evaluated as describedabove.

EXAMPLE 7 Assay of Modulator Compound Stability in Cerebrospinal Fluid

[0205] The stability of a modulator compound in cerebrospinal fluid(CSF) can be assayed in an in vitro assay as follows. A CSF solution isprepared containing 75% Rhesus monkey CSF (commercially available fromNorthern Biomedical Research), 23% sterile phosphate buffered saline and2% dimethylsulfoxide (v/v)(Aldrich Chemical Co., Catalog No. 27,685-5).Test modulator compounds are added to the CSF solution to a finalconcentration of 40 μM or 15 μM. All sample handling is carried out in alaminar flow hood and test solutions are maintained at 37° C. during theassay. After 24 hours, enzymatic activity in the solutions is quenchedby adding acetonitrile to produce a final concentration of 25% (v/v).Samples (at the 0 time point and the 24 hour time point) are analyzed atroom temperature using reverse-phase HPLC. A microbore column is used tomaximize sensitivity. The parameters for analytical HPLC are as follows:

[0206] Solvent System

[0207] A: 0.1% Trifluoroacetic acid (TFA) in water (v/v)

[0208] B: 0.085% TFA/Acetonitrile, 1% H₂O (v/v)

[0209] Injection and Gradient

[0210] Inject: 100-250 μL of test sample

[0211] Run: 10% for B for 5 min., then 10-70% B over 60 min.

[0212] Chromatographic analysis is performed using a Hewlett Packard1090 series II HPLC. The column used for separation is a C4, 5 μm, 1×250mm (Vydac #214TP51). The flow rate is 50 μL/min and the elution profileof the test compounds is monitored at 214, 230, 260 and 280 nm.

EXAMPLE 8 Brain Uptake Assay

[0213] Brain levels of our Aβ-derived peptides were determined in therat following intervenous administration. Under ketamine/xylazineanesthesia male Sprague-Dawley rats (219-302g) received an intravenousinjection via a catheter inserted in the left jugular vein (dose volumeof 4 mL/kg administered over 1 minute) The actual dose administered ofeach compound tested is shown in FIG. 1.

[0214] At 60 minutes post administration the left common carotid arterywas cannulated to enable perfusion of the left forebrain to removecerebral blood. The left forebrain, void of blood was subjected tocapillary depletion as described by (Triguero et al. (1990) J.Neurochem. 54:1882-1888). This established technique separates brainvasculature from the parenchyma and, thus, allows the accuratedetermination of the concentration of compound under investigation thathas traversed the blood brain barrier. The amount of parent compoundthat was present within the brain was determined by LC/MS/MS.

[0215] The above-described assay was used to measure the brain uptake ofthe modulators: Com- Dose pounds Conc mg/kg PPI Structure mwt (mg/mL) IV1324 TFA. H-(l-[F₅]f-fvl)-NH2 841 1.20 4.9 1318 TFA. H-(lf-D-Cha-vl)-NH2757 0.29 1.0 1319 TFA. H-(lf-[p-F]f-vl)-NH2 769 1.70 6.6 1327 TFA.H-(l-[p-F]f-[p-F]f-vl)-NH2 787 0.98 4.0 1301 TFA. H-(lvf-D-Cha-l)-NH2757 0.70 2.9 1302 TFA. H-(lvf-[p-F]f-l)-NH2 769 0.19 0.7 1328 TFA.H-(l-[F₅]f-[F₅]f-vl)-NH2 931 0.29 1.2 1322 TFA. H-(l-D-Cha-fvl)-NH2 7570.03 0.1 1303 TFA. H-(lvf-[F₅]f-l)-NH2 841 0.27 1.0 1326 TFA.H-(l-D-Cha-D-Cha-vl)- 763 0.05 0.2 NH2 1320 TFA. H-(lf-[F₅]f-vl)-NH2 8410.70 3.0

[0216] The β-amyloid modulator compounds described herein are summarizedin the following Table. TABLE IV PPI# Description SEQ ID NO  803 TFA •N,N-dimethyl-(Gaffvl)-NH₂  913 TFA • N,N-dimethyl-(affvl)-NH₂  918 TFA •H-(l-[Me]v-ffa)-NH₂  968 TFA • N-methyl-(Gaffvl)-NH₂  969 TFA •N-ethyl-(Gaffvl)-NH₂  970 TFA • N-isopropyl-(Gaffvl)-NH₂  992 TFA •H-(lvffa)-isopropylamide  993 TFA • H-(lvffa)-dimethylamide 1005 TFA •N,N-diethyl-(Gaffvl)-NH₂ 1006 TFA • N,N-diethyl-(affvl)-NH₂ 1007 TFA •N,N-dimethyl-(lvffl)-NH₂ 1008 TFA • N,N-dimethyl-(lffvl)-NH₂ 1013 TFA •H-(Glvffl)-NH₂ 1017 TFA • N-ethyl-(Glvffl)-NH₂ 1018 TFA •N-ethyl-(Glffvl)-NH₂ 1020 TFA • N-methyl-(lffvl)-NH₂ 1022 TFA •N-ethyl-(lvffl)-NH₂ 1025 TFA • N-propyl-(lvffl)-NH₂ 1028 TFA •N,N-diethyl-(Glvffl)-NH₂ 1038 TFA • H-(ivffi)-NH₂ 1039 TFA •H-(ivffa)-NH₂ 1040 TFA • H-(iiffi)-NH₂ 1041 TFA • H-(D-Nle-vffa)-NH₂1042 TFA • H-(D-Nle-vff-D-Nle)-NH₂ 1088 TFA •1-piperidine-acetyl-(lvffl)-NH₂ 1089 TFA •1-piperidine-acetyl-(lffvl)-NH₂ 1093 TFA • H-lvffl-isopropylamide 1094TFA • H-lffvl-isopropylamide 1179 TFA • H-(lvffl)-methylamide 1180 TFA •H-(lffvl)-methylamide 1261 TFA • H-(lvffl)-OH 1019 TFA •N-methyl-(lvffl)-NH₂ 1301 TFA • H-(lvf-D-Cha-l)-NH₂ 1302 TFA •H-(lvf-[p-F]f-l)-NH₂ 1303 TFA • H-(lvf-[F₅]f-1)-NH₂ 1306N-methyl-(lvf-D-Cha-l)-NH₂ 1307 N-methyl-(lvf-[p-F]f-l)-NH₂ 1308N-methyl-(lvf-[F₅]f-l)-NH₂ 1318 TFA • H-(lf-D-Cha-vl)-NH₂ 1319 TFA •H-(lf-[p-F]f-vl)-NH₂ 1320 TFA • H-(lf-[F₅]f-vl)-NH₂ 1321 2TFA •H-(lfkvl)-NH₂ 1322 TFA • H-(l-D-Cha-fvl)-NH₂ 1323 TFA •H-(l-[p-F]f-fvl)-NH₂ 1324 TFA • H-(l-[F₅]f-fvl)-NH₂ 1325 2TFA •H-(lkfvl)-NH₂ 1326 TFA • H-(l-D-Cha-D-Cha-vl)-NH₂ 1327 TFA •H-(l-[p-F]f-[p-F]f-vl)-NH₂ 1328 TFA • H-(l-[F₅]f-[F₅]f-vl)-NH₂ 1329 3TFA • H-(lkkvl)-NH₂ 1125 2 TFA • H-lvf-NH—NH-fvl-H 1133 TFA •H-lvf-NH—NH-Acetyl 1155 TFA • H-lvf-NH—NH₂

[0217] Equivalents

[0218] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 25 1 43 PRT Artificial Sequence Polypeptide 1 Asp Ala Glu Phe Arg HisAsp Ser Gly Tyr Gly Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe AlaGlu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val GlyGly Val Val Ile Ala Thr 35 40 2 103 PRT Artificial Sequence Polypeptide2 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 1 5 1015 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 20 2530 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 35 4045 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile 50 5560 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 65 7075 80 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 8590 95 Phe Phe Glu Gln Met Gln Asn 100 3 4 PRT Artificial SequencePolypeptide 3 Leu Val Phe Phe 1 4 5 PRT Artificial Sequence Polypeptide4 Leu Val Phe Phe Ala 1 5 5 5 PRT Artificial Sequence Polypeptide 5 LeuVal Phe Xaa Leu 1 5 6 5 PRT Artificial Sequence Polypeptide 6 Leu ValXaa Phe Leu 1 5 7 5 PRT Artificial Sequence Polypeptide 7 Leu Val PhePhe Leu 1 5 8 5 PRT Artificial Sequence Polypeptide 8 Leu Val Phe PheLeu 1 5 9 5 PRT Artificial Sequence Polypeptide 9 Leu Val Phe Phe Leu 15 10 5 PRT Artificial Sequence Polypeptide 10 Leu Val Phe Phe Leu 1 5 115 PRT Artificial Sequence Polypeptide 11 Leu Phe Xaa Val Leu 1 5 12 5PRT Artificial Sequence Polypeptide 12 Leu Phe Phe Val Leu 1 5 13 5 PRTArtificial Sequence Polypeptide 13 Leu Phe Phe Val Leu 1 5 14 5 PRTArtificial Sequence Polypeptide 14 Leu Phe Lys Val Leu 1 5 15 5 PRTArtificial Sequence Polypeptide 15 Leu Xaa Phe Val Leu 1 5 16 5 PRTArtificial Sequence Polypeptide 16 Leu Phe Phe Val Leu 1 5 17 5 PRTArtificial Sequence Polypeptide 17 Leu Phe Phe Val Leu 1 5 18 5 PRTArtificial Sequence Polypeptide 18 Leu Lys Phe Val Leu 1 5 19 5 PRTArtificial Sequence Polypeptide 19 Leu Xaa Xaa Val Leu 1 5 20 5 PRTArtificial Sequence Polypeptide 20 Leu Val Xaa Xaa Leu 1 5 21 5 PRTArtificial Sequence Polypeptide 21 Leu Phe Phe Val Leu 1 5 22 5 PRTArtificial Sequence Polypeptide 22 Leu Val Phe Phe Leu 1 5 23 5 PRTArtificial Sequence Polypeptide 23 Leu Phe Phe Val Leu 1 5 24 5 PRTArtificial Sequence Polypeptide 24 Leu Val Phe Phe Leu 1 5 25 3 PRTArtificial Sequence Polypeptide 25 Leu Val Phe 1

We claim:
 1. A compound comprising the structure:

wherein Xaa₁and Xaa₂ are each D-amino acid structures and at least twoof Xaa₁and Xaa₂ are, independently, selected from the group consistingof a D-leucine structure, a D-phenylalanine structure, a D-tyrosinestructure, a D-iodotyrosine structure, a D-lysine structure, or aD-valine structure; NH—NH is a hydrazine structure; Y, which may or maynot be present, is a structure having the formula (Xaa)_(a), wherein Xaais any D-amino acid structure and a is an integer from 1 to 15; Xaa₁′,Xaa₂′, and Xaa₃′ which may or may not be present, are each D-amino acidor L-amino acid structures and at least two of Xaa₁′, Xaa₂′, and Xaa₃′are, independently, selected from the group consisting of a D- orL-leucine structure, a D- or L-phenylalanine structure, a D- orL-tyrosine structure, a D- or L-iodotyrosine structure, a D- or L-lysinestructure, or a D- or L-valine structure; Z, which may or may not bepresent, is a structure having the formula (Xaa)_(b), wherein Xaa is anyD-amino acid structure and b is an integer from 1 to 15; A, which may ormay not be present, is a modifying group attached directly or indirectlyto the compound; and n is an integer from 1 to 15; wherein Xaa₁, Xaa₂,Xaa₁′, Xaa₂′, Xaa₃′, Y, Z, A and n are selected such that the compoundbinds to natural β-amyloid peptides or modulates the aggregation orinhibits the neurotoxicity of natural β-amyloid peptides when contactedwith the natural β-amyloid peptides, and is less prone to metabolism. 2.A compound having a structure selected from the group consisting of:H-D-Leu-D-Val-D-Phe-NH-(H-D-Leu-D-Val-D-Phe-)NH;H-D-Leu-D-Val-D-Phe-NH-NH-COCH₃; and H-D-Leu-D-Val-D-Phe-NH-NH₂.
 3. Acompound comprising the structure:

wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are each D-amino acid structures andat least two of Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are, independently, selectedfrom the group consisting of a D-leucine structure, aD-cyclohexylalanine, a D-4-fluorophenylalanine(para-fluorophenylalanine), a D-pentafluorophenylalanine, achlorophenylalanine, a bromophenylalanine, a nitrophenylalanine, aD-homophenylalanine, a D-lysine structure, and a D-valine structure; Y,which may or may not be present, is a structure having the formula(Xaa)_(a), wherein Xaa is any D-amino acid structure and a is an integerfrom 1 to 15; Z, which may or may not be present, is a structure havingthe formula (Xaa)_(b), wherein Xaa is any D-amino acid structure and bis an integer from 1 to 15; A, which may or may not be present, is amodifying group attached directly or indirectly to the compound; and nis an integer from 1 to 15; wherein Xaa₁, Xaa₂, Xaa₃, Xaa₄, Y, Z, A andn are selected such that the compound binds to natural β-amyloidpeptides or modulates the aggregation or inhibits the neurotoxicity ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides, and is less prone to metabolism.
 4. A compound having astructure selected from the group consisting of:N,N-dimethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-dimethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-methyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-ethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-isopropyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-isopropylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-dimethylamide;N,N-diethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-diethyl-(D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-dimethyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-ethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-ethyl-(Gly-D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-methyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;N-ethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N-propyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;N,N-diethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ile)-NH₂;H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ala-)-NH₂;H-(D-Ile-D-Ile-D-Phe-D-Phe-D-Ile)-NH₂;H-(D-Nle-D-Val-D-Phe-D-Phe-D-Ala-)-NH₂;H-(D-Nle-D-Val-D-Phe-D-Phe-D-Nle)-NH₂;1-piperidine-acetyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;1-piperidine-acetyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH₂;H-D-Leu-D-Val-D-Phe-D-Phe-D-Leu-isopropylamide;H-D-Leu-D-Phe-D-Phe-D-Val-D-Leu-isopropylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-methylamide;H-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-methylamide;H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-OH;N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH₂;H-(D-Leu-D-Val-D-Phe-D-[F₅]Phe-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-Cha-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-[p-F]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-[F₅]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Phe-D-Lys-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Cha-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[p-F]Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[F₅]Phe-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Lys-D-Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Cha-D-Cha-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[p-F]Phe-D-[p-F]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-[F₅]Phe-D-[F₅]Phe-D-Val-D-Leu)-NH₂;H-(D-Leu-D-Lys-D-Lys-D-Val-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH₂;N-methyl-(D-Leu-D-Val-D-Phe-D-[F₅]Phe-D-Leu)-NH₂;H-D-Leu-D-Val-D-Phe-NH-(H-D-Leu-D-Val-D-Phe-)NH;H-D-Leu-D-Val-D-Phe-NH—NH—COCH₃; H-D-Leu-D-Val-D-Phe-NH—NH₂H-(lv-[3-I]y-fa)-NH₂; H-(lvffl)-NH-Et; H-lvffl-NH-CH₂CH₂-NH₂;H-(GGClvffl)-NH₂; H-(GGClvfyl)-NH₂; H-(GGClvf-[3-I]y-l)-NH₂;H-LVF-NH—-NH-FVL-H; H-LVF-NH—NH-fvl-H; H-lff-(nvl)-1-NH₂;H-lf-[pF]f-(nvl)-l-NH₂; H-l-[pF]f-[pF]f-(nvl)-1-NH₂; Me-lvyfl-NH₂;H-(lvyfl)-NH₂; Me-(lv-[p-F]f-fl)-NH₂; H-(lv-[p-F]f-fl)-NH₂;H-(lvf-[p-F]f-l)-NH₂; lvff-[nvl])-NH₂; Me-(lvff-[nle])-NH₂;Me-(lvffl)-OH; Me-(lvffl)-NH—OH; H-(lv-[p-F]f-f-(nvl))-NH₂;Me-(l-v-[p-F]f-f-(nvl))-NH₂; H-((nvl)-v-[p-F]f-f-nvl)-NH₂;H-(l-(nvl)-[p-F]f-f-(nvl)-NH₂; H-((nvl)-(nvl)-[p-F]f-f-(nvl))-NH₂;Me-(l-(nvl)-[p-F]f-f-(nvl))-NH₂; H-(lvff-(nvl))-NH₂; Ac-(lvffl)-NH₂;Ac-(lvffl)-OH; and H-(lv-[3-I]y-fl)-NH₂.
 5. A compound having thestructure: H-(D-Leu-D-Phe-[p-F]D-Phe-D-Val-D-Leu)-NH₂.
 6. A compoundhaving the structure: N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH₂.
 7. Apharmaceutical composition comprising a therapeutically effective amountof the compound of any of claims 1, 2, 3, 4, 5, or 6 and apharmaceutically acceptable carrier.
 8. A method for inhibitingaggregation of natural β-amyloid peptides, comprising contacting thenatural β-amyloid peptides with the compound of any of claims 1, 2, 3,4, 5, or 6 such that aggregation of the natural β-amyloid peptides isinhibited.
 9. A method for detecting the presence or absence of naturalβ-amyloid peptides in a biological sample, comprising: contacting abiological sample with the compound of any of claims 1, 2, 3, 4, 5, or6, wherein the compound is labeled with a detectable substance; anddetecting the compound bound to natural β-amyloid peptides to therebydetect the presence or absence of natural β-amyloid peptides in thebiological sample.
 10. The method of claim 9, wherein the β-amyloidmodulator compound and the biological sample are contacted in vitro. 11.The method of claim 9, wherein the β-amyloid modulator compound iscontacted with the biological sample by administering the β-amyloidmodulator compound to a subject.
 12. The method of claim 9, wherein thecompound is labeled with radioactive technetium or radioactive iodine.13. A method for treating a subject for a disorder associated withβ-amyloidosis, comprising: administering to the subject atherapeutically effective amount of the compound of any of claims 1, 2,3, 4, 5, or 6 such that the subject is treated for a disorder associatedwith β-amyloidosis.
 14. The method of claim 13, wherein the disorder isAlzheimer's disease.